Synthesis of 3H- and 2H4-labelled versions of the hypoxia-activated pre-prodrug 2-((2-bromoethyl)-2,4-dinitro-6-(((2- (phosphonooxy)ethyl)amino)carbonyl)anilino)ethyl methanesulfonate (PR-104)

Article abstract of DOI:10.1002/jlcr.1147

³H- and ²H₄-versions of the hypoxia-activated pre-prodrug PR-104 [2-[(2-bromoethyl)-2,4-dinitro-6-[[[2- (phosphonooxy)ethyl]amino]carbonyl]anilino]ethyl methanesulfonate], labelled in the ethylcarboxamide side chain, have

Full text of DOI:10.1002/jlcr.1147

Journal of Labelled Compounds and Radiopharmaceuticals
J Label Compd Radiopharm 2007; 50: 7–12.
Published online in Wiley InterScience
JLCR
(www.interscience.wiley.com). DOI: 10.1002/jlcr.1147
Research Article
3
2
Synthesis of H- and H4-labelled versions of the hypoxia-
activated pre-prodrug 2-[(2-bromoethyl)-2,4-dinitro-6-
[[[2-(phosphonooxy)ethyl]amino]carbonyl]anilino]ethyl
methanesulfonate (PR-104)
GRAHAM J. ATWELL and WILLIAM A. DENNY*
Auckland Cancer Society Research Centre, School of Medical Sciences, The University of Auckland, Private Bag 92019, Auckland Mail Centre,
Auckland 1142, New Zealand
Received 25 September 2006; Revised 2 October 2006; Accepted 3 October 2006
Abstract: 3H- and 2H4-versions of the hypoxia-activated pre-prodrug PR-104 [2-[(2-bromoethyl)-2,4-dinitro-6-[[[2-
(phosphonooxy)ethyl]amino]carbonyl]anilino]ethyl methanesulfonate], labelled in the ethylcarboxamide side chain,
have been prepared, respectively, by [3H]NaBH4 reduction of a precursor late stage aldehyde, and by late stage
incorporation of deuterium with 2-amino[1,1,2,2-2H4]ethanol. Copyright # 2007 John Wiley & Sons, Ltd.
Keywords: dinitrobenzamide mustard; hypoxia-activated prodrug; PR-104; tritium; deuterium
Introduction
intermediate. A stably labelled version of 1 was also
required, to use as an internal standard for the mass
spectrometric analysis of preclinical and clinical plas-
ma samples. The presence of natural isotopes of the
bromine in 1 necessitated a standard that differed by at
least three mass units, and a synthesis that exploited
the late stage introduction of commercially available
tetra-deutero 2-aminoethanol was devised.
The asymmetric mustard dinitrobenzamide phosphate
PR-104 (1)1,2 is a soluble pre-prodrug designed to be
converted rapidly into the prodrug 2 by serum phos-
phatases (Scheme 1). This alcohol then diffuses
efficiently to, and is activated selectively in, hypoxic
regions of solid tumors3 to reactive reduced species
(e.g. 3 and 4) that are cytotoxic DNA cross-linking
agents.4 PR-104 shows excellent in vivo activity against
the subfraction of hypoxic cells in human tumor
xenografts,1,2 and has recently begun Phase I clinical
trials.
Discussion
The synthesis of tritium-labelled 1 (1-T) is shown in
Scheme 2. A number of oxidizing agents were evaluated
for the conversion of alcohol 2 to aldehyde 5, the best
being Dess-Martin periodinane and the eventually
employed pyridinium chlorochromate (PCC). Reduction
of 5, used in excess) with [3H]NaBH4 (60 Ci/mmol,
diluted two-fold with unlabelled NaBH4) in ethanol/
THF at 208C for 20 min gave the labelled alcohol 6 in
good yield, with a measured specific activity of 6.3 Ci/
mmol. Compound 6 (diluted 10-fold with unlabelled 2)
was phosphorylated as previously reported1 for 2, with
di-tert-butyl diisopropylphosphoramidite/1H-tetrazole
followed by oxidation with 70% hydrogen peroxide, to
provide the phosphate ester 7. This was cleaved with
trifluoroacetic acid to give phosphate diacid 1-T in 65%
overall yield from 6, with a measured specific activity of
0.77 Ci/mmol.
As part of the preclinical development of 1, a tritium-
labelled version was required for drug disposition and
metabolic studies. As previous work4 had shown that
oxidative loss of one or both arms of the mustard was a
minor pathway in the metabolism of compounds
related to 1, it was decided to place the label in the
carboxamide side chain. This had the additional benefit
of being able to start with prodrug 2 as the late stage
*Correspondence to: William A. Denny, Auckland Cancer Society
Research Centre, School of Medicine Sciences, The University of
Auckland, Private Bag 92019, Auckland Mail Centre, Auckland,
1142, New Zealand. E-mail: b.denny@auckland.ac.nz
Contract/grant sponsor: Health Research Council of New Zealand;
contract/grant number: 01/276
Contract/grant sponsor: Proacta Therapeutics Ltd
Copyright # 2007 John Wiley & Sons, Ltd.

8
G. J. ATWELL AND W. A. DENNY
NHOH
NO2
NH2
ii
+
H
N
H
N
H
N
O2N
Br
OH
O2N
R
O2N
OH
N
O
N
O
N
O
OMs
3
Br
i
OMs
4
Br
OMs
1: R=OP(O)(OH)2
2: R=OH
(i) Serum phosphatases; (ii) cellular reductases under hypoxia.
Scheme 1 Mechanism of activation of PR-104.
NO2
NO2
NO2
i
ii
T
H
N
H
N
H
N
CHO
O2N
Br
OH
OH
O2N
Br
O2N
Br
N
O
N
O
N
O
OMs
OMs
OMs
5
6
2
NO2
NO2
T
T
H
N
iv
H
iii
N
OP(O)(OtBu)2
O2N
O2N
X
OP(O)(OH)2
N
O
N
O
X
OMs
OMs
1-T
7
Reagents: (i)PCC; (ii) [T]NaBH4/EtOH; (iii) i-Pr2NP(OtBu)2/1H-tetrazole, then
H2O2; (iv) TFA/CH2Cl2
Scheme 2 Synthesis of 3H PR-104.
For analysis and quantitation of the preclinical
and clinical plasma samples from treatment with 1 by
mass spectrometry, an internal standard, differing
by at least three mass units, was required. The
commercial availability of tetra-deuterated 2-amino-
ethanol allowed the opportunity of incorporating
four D atoms in the carboxamide side chain to give
1-D4. Rather than repeat the standard synthesis,1
which involved introduction of the side chain early
in the synthesis, an alternative route was devised
where the side chain was incorporated as late as
possible (after elaboration of the mustard group)
(Scheme 3).
unreacted 10 by column chromatography. The ester 11
was then cleaved with trifluoroacetic acid to give the
key acid 12 in 33% overall yield from 8.
Activation of acid 12 with a number of coupling
reagents (DECP, CDI, EDCI) proved unsuccessful.
However, conversion of 12 to the corresponding
acid chloride (oxalyl chloride, catalytic DMF)
followed by reaction with 2-amino[1,1,2,2-2H4]ethanol
in the presence of triethylamine gave the labelled
alcohol 13 in good yield. HPLC analysis showed this
contained 4.4% of the corresponding chloromesylate,
generated by chloride exchange of the bromide. The
alcohol 13 was phosphorylated with di-tert-butyl
In this procedure, the known5 tert-butyl ester 8 was
reacted with diethanolamine to give the diol 9, which
was converted to the dimesylate 10 with mesyl chloride
in pyridine. This was treated with lithium bromide in
DMF, and the resulting bromomesylate 11 was sepa-
rated from the corresponding dibromo compound and
diisopropylphosphoramidite/1H-tetrazole
as
pre-
viously described1 for 2, and the resulting phosphate
ester 14 was cleaved with trifluoroacetic acid to give
phosphate diacid 1-D4, in 16% overall yield from 8.
Deuterium incorporation (98%) was confirmed by high
resolution FAB mass spectrometry.
Copyright # 2007 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 7–12
DOI: 10.1002.jlcr

SYNTHESIS OF 3H- AND 2H4-LABELLED VERSIONS OF THE HYPOXIA-ACTIVATED PRE-PRODRUG
9
NO2
NO2
NO2
NO2
Cl
i
v
iii
D
D
H
N
D
D
t
t
OH
CO2R
OMs
O2N
Br
O2N
O2N
O2N
CO2 Bu
CO2 Bu
N
O
N
N
OMs
Br
iv
R
ii
R
8
13
11: R=tBu
9: R=OH
12: R=H
10: R=OMs
NO2
NO2
N
vii
vi
D
D
D
H
D
H
D
N
N
O2N
Br
OP(O)(OtBu)2
OP(O)(OH)2
O2N
Br
D
N
O
D
O
D
OMs
OMs
14
1-D4
Reagents: (i) HN(CH2)CH2OH)2; (ii) MsCl; (iii) LiBr; (iv) TFA; (v) (COCl)2, then
H2N(CD2)2OH; (vi) i-Pr2NP(OtBu)2/1H-tetrazole, then H2O2; (vii) TFA/CH2Cl2
Scheme 3 Synthesis of 2H4 PR-104.
(50 ml) and filtered. The organic phase was washed
with water (50 ml), dried and concentrated under
reduced pressure. The residue was chromatographed
on silica gel, eluting with EtOAc/petroleum ether (4:1).
The middle fractions were combined and rechromato-
graphed, eluting with EtOAc/petroleum ether (6:1) to
give 5 (413 mg, 69%) as a yellow gum which was used
directly: 1H NMR [(CD3)2SO] d 9.61 (s, 1H), 9.15
(t, J ¼ 5:4 Hz, 1H), 8.77 (d, J ¼ 2:8 Hz, 1H), 8.42
(d, J ¼ 2:8 Hz, 1H), 4.28 (t, J ¼ 5:4 Hz, 2H), 4.20
(t, J ¼ 5:5 Hz, 2H), 3.62–3.54 (m, 2H), 3.54–3.43
(m, 4H), 3.12 (s, 3H).
Experimental section
Analyses were performed by the Microchemical La-
boratory, University of Otago, Dunedin, NZ. Melting
points were determined using an Electrothermal Model
9200 digital melting point apparatus, and are as read.
NMR spectra were measured on a Bruker DRX–400
spectrometer, and referenced to Me4Si. Mass spectra
were recorded on a Varian VG-70SE spectrometer.
HPLC was carried out using a Bondclone 10 C18
reverse-phase silica gel column, with
a Phillips
PU4100 M gradient elution pump and a Phillips PU
4120 diode array detector, and eluting with the
appropriate ratios of 95% MeCN/5% water (solvent A)
and 0.45 M ammonium formate buffer (solvent B; pH
[3H]2-[(2-Bromoethyl)-2-[[(2-hydroxyethyl)amino]-
carbonyl]-4,6-dinitroanilino]ethyl methanesulfonate
(6)
3.45). The specific activities of
6 and 1-T were
determined by quantitation of 3H-labelled samples by
HPLC, and subsequent off-line liquid scintillation
counting (Packard 1500 Tri-Carb) of the isolated HPLC
peaks.
A freshly prepared solution of [3H]NaBH4 (0.84 mg,
0.02 mmol, nominally 1.2 Ci at 60 Ci/mmol) (Amer-
sham Biosciences, UK) and unlabelled NaBH4
(0.76 mg, 0.02 mmol) in EtOH (1.5 ml) was added
dropwise to a stirred solution of 5 (99 mg, 0.20 mmol)
in THF (5 ml) at 208C over a period of 5 min. The
mixture was stirred at 208C for a further 15 min, then
quenched with 0.1 N aqueous MsOH (1 ml), diluted with
water (20 ml) and extracted with EtOAc (2 ꢀ 20 ml). The
combined organic layer was washed with water
(2 ꢀ 20 ml), dried, and concentrated under reduced
pressure. Chromatography on silica gel, eluting with
EtOAc/petroleum ether (6:1), gave unreacted 5 (8 mg),
then 6 (60 mg, 75%); (6.3 Ci/mmol) as a yellow gum,
identical on HPLC with unlabelled 2: 1H NMR
[(CD3)2SO] d 8.76–8.70 (m, 2H), 8.35 (d, J ¼ 2:8 Hz,
2-[(2-Bromoethyl)-2,4-dinitro-6-[[(2-oxoethyl)amino]-
carbonyl]anilino]ethyl methanesulfonate (5)
A solution of 2-[(2-bromoethyl)-2-[[(2-hydroxyethyl)a-
mino]carbonyl]-4,6-dinitroanilino]ethyl methanesulfo-
nate1 (2) (600 mg, 1.20 mmol) in dry CH2Cl2 (125 ml)
was stirred with 3A powdered molecular sieves (1.5 g)
for 5 min at room temperature, then treated with
powdered
pyridinium
chlorochromate
(777 mg,
3.60 mmol). The mixture was stirred at room tempera-
ture for a further 15 min, then treated with water
Copyright # 2007 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 7–12
DOI: 10.1002.jlcr

10 G. J. ATWELL AND W. A. DENNY
1H), 4.83–4.75 (m. 1H), 4.28 (t, J ¼ 5:5 Hz, 2H), 3.61–
3.52 (m, 4H), 3.51–3.42 (m, 4H), 3.39–3.31 (m, 2H),
3.13 (s, 3H).
treated with diethanolamine (2.84 g, 27 mmol) and
vigorously stirred at room temperature for 3 h. Dilution
with water provided a solid which was purified by
column chromatography on silica gel, eluting with
EtOAc/petroleum ether (4:1), followed by recrystalliza-
tion from EtOAc/iPr2O to give tert-butyl 2-[bis(2-
hydroxyethyl)amino]-3,5-dinitrobenzoate (9) (3.78 g,
94%) as a yellow solid: m.p. 117–1188C. 1H NMR
[3H]2-[(2-Bromoethyl)-2-(6-tert-butoxy-8,8-dimethyl-
6-oxido-5,7-dioxa-2-aza-6-phosphanon-1-anoyl)-
4,6-dinitroanilino]ethyl methanesulfonate (7)
[(CD3)2SO]
d 8.69 (d, J ¼ 2:8 Hz, 1H), 8.40 (d,
A stirred solution of 6 (30 mg, 0.06 mmol; 235 GBq/
J ¼ 2:8 Hz, 1H), 4.53 (t, J ¼ 5:6 Hz, 2H), 3.57 (q,
J ¼ 5:9 Hz, 4H), 3.19 (t, J ¼ 6:1 Hz, 4H), 1.60
mmol [6.3 Ci/mmol] and unlabelled
2
(270 mg,
0.54 mmol) in dry DMF (1.5 ml) at 108C was treated
with 1H-tetrazole (67 mg, 0.96 mmol), followed by the
dropwise addition of di-tert-butyl diisopropylpho-
sphoramidite (0.26 ml, 0.78 mmol), 95%). The mixture
was warmed to room temperature of 2 h, then cooled to
08C and treated slowly with a solution of 70% aqueous
H2O2 (0.15 ml) in THF (0.2 ml). The mixture was stirred
at 108C for a further 30 min, then poured into cold
water (25 ml) and extracted with EtOAc (2 ꢀ 10 ml). The
combined organic layers were washed with 5% aqueous
Na2S2O5 (10 ml), water (4 ꢀ 10 ml), dried, and evapo-
rated under reduced pressure. The residue was chro-
matographed on silica gel, eluting with EtOAc/
petroleum ether, and the eluant was concentrated
under reduced pressure below 308C to a small volume
and diluted with hexane to precipitate 7 (303 mg, 73%)
as a yellow oil: 1H NMR [(CD3)2SO] d 8.93 (t, J ¼ 5:6 Hz,
1H), 8.75 (d, J ¼ 2:8 Hz, 1H), 8.34 (d, J ¼ 2:8 Hz, 1H),
4.28 (t, J ¼ 5:4 Hz, 2H), 4.06–3.97 (m, 2H), 3.61–3.43
(m, 8H), 3.13 (s, 3H), 1.43 (s, 18H).
(s, 9 H). Analytically required for
C15H21N3O8: C,
48.52; H, 5.7; N, 11.32. Found: C, 48.54; H, 5.8; N,
11.31.
tert-Butyl 2-[bis[2-[(methylsulfonyl)oxy]ethyl]amino]-
3,5-dinitrobenzoate (10)
A stirred solution of 9 (3.76 g, 10.1 mmol) in pyridine
(5 ml) at 08C was treated dropwise with MsCl (1.90 ml,
24.5 mmol), and then allowed to come to room tem-
perature for 1 h. The mixture was diluted with water
(50 ml) and the resulting solid was purified by column
chromatography on silica gel, eluting with EtOAc,
followed by recrystallization from EtOAc/iPr2O to give
10 (4.79 g, 90%) as a yellow solid: m.p. 105.5–1068C.
1H NMR [(CD3)2SO] d 8.81 (d, J ¼ 2:8 Hz, 1H), 8.53
(d, J ¼ 2:8 Hz, 1H), 4.30 (t, J ¼ 5:5 Hz, 4H), 3.49
(t, J ¼ 5:5 Hz, 4H), 3.13 (s, 6H), 1.61 (s, 9 H). Analyti-
cally required for C17H25N3O12S2: C, 38.71; H, 4.78; N,
7.97; S, 12.16. Found: C, 38.78; H, 4.82; N, 7.96; S,
12.30.
[3H]2-[(2-Bromoethyl)-2,4-dinitro-6-[[[2-(phospho-
nooxy)ethyl]-amino]carbonyl]anilino]ethyl metha-
nesufonate (1-T)
tert-Butyl 2-((2-bromoethyl)-(2-[(methylsulfonyl)oxy]
ethyl]amino]-3,5-dinitrobenzoate (11)
A solution of 7 (299 mg, 0.43 mmol) in CH2Cl2 (2 ml)
was treated with TFA (2 ml) at room temperature for 1 h,
then concentrated to near dryness under reduced
pressure below 308C. The residual gum was dissolved
in dry MeCN (0.2 ml) and the solution was diluted with
dry CH2Cl2 (10 ml), filtered, seeded, and refrigerated at
58C for 16 h. The separated solid was collected, washed
with CH2Cl2 and dried under high vacuum to give 1-T
(223 mg, 89%; 98.5% pure by HPLC) (0.77 Ci/mmol) as
yellow crystals. 1H NMR [(CD3)2SO] d 11.0 (br, 2H),
8.97–8.90 (m, 1H), 8.74 (d, J ¼ 2:8 Hz, 1H), 8.37 (d,
J ¼ 2:8 Hz, 1H), 4.29 (t, J ¼ 5:4 Hz, 2H), 3.98 (q,
J ¼ 6:2 Hz, 2H), 3.62–3.43 (m, 8H), 3.13 (s, 3H).
A mixture of 10 (5.00 g, 9.48 mmol) and LiBr (0.99 g,
11.4 mmol) in DMF (10 ml) was stirred at 508C for 1.5 h,
then diluted with water and refrigerated. The precipi-
tated semi-solid was dried and chromatographed on
silica gel, eluting with EtOAc/petroleum ether (9:11) to
give firstly tert-butyl 2-[bis(2-bromoethyl)amino]-3,
5-dinitrobenzoate (1.60 g) as a yellow solid: 1H NMR
[(CD3)2SO]
d
8.80 (d, J ¼ 2:8 Hz, 1H), 8.50
(d, J ¼ 2:8 Hz, 1H), 3.61–3.54 (m, 4H), 3.54–3.47
(m, 4H), 1.61 (s, 9 H).
Later fractions gave 11 (2.11 g, 43%) as a yellow
solid: m.p. (EtOAc/iPr2O) 97–988C. 1H NMR [(CD3)2SO]
d 8.81 (d, J ¼ 2:8 Hz, 1H), 8.52 (t, J ¼ 2:8 Hz, 1H), 4.29
(t, J ¼ 5:5 Hz, 2H), 3.62–3.55 (m, 2H), 3.54–3.46 (m,
4H), 3.12 (s, 3H), 1.60 (s, 9 H). Analytically required for
tert-Butyl 2-[bis(2-hydroxyethyl)amino]-3,5-dinitro-
benzoate (9)
A solution of tert-butyl 2-chloro-3,5-dinitrobenzoate5
(8) (3.27 g, 10.8 mmol) in dioxane (10 ml) at 108C was
C
16H22BrN3O9S: C, 37.51; H, 4.33; N, 8.2; Br, 15.60.
Found: C, 37.65; H, 4.51; N, 8.12; Br, 15.69.
Copyright # 2007 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 7–12
DOI: 10.1002.jlcr

SYNTHESIS OF 3H- AND 2H4-LABELLED VERSIONS OF THE HYPOXIA-ACTIVATED PRE-PRODRUG 11
2-[(2-Bromoethyl)-2-([3,3,4,4-2H4]-6-tert-butoxy-8,
8-dimethyl-6-oxido-5,7-dioxa-2-aza-6-phosphanon-
1-anoyl)-4,6-dinitroanilino]ethyl methanesulfonate
(14)
Further elution with EtOAc gave unreacted 10
(0.92 g).
2-[(2-Bromoethyl)-(2-[(methylsulfonyl)oxy]ethyl]
amino]-3,5-dinitrobenzoic acid (12)
A stirred solution of 13 (1.55 g, 3.08 mmol) and 1H-
tetrazole (345 mg, 4.93 mmol) in dry DMF (8 ml) at 108C
was treated dropwise with di-tert-butyl diisopropylpho-
sphoramidite (1.33 ml, 4.00 mmol, 95%). The mixture
was warmed at room temperature for 2 h, then cooled
to 08C and treated slowly with a solution of 70%
aqueous H2O2 (0.8 ml) in THF (1 ml). The mixture was
warmed to 108C for 0.5 h, then poured into ice-cold
water (100 ml) and extracted with EtOAc (2 ꢀ 40 ml).
The combined organic phases were washed with 5%
aqueous Na2S2O5 (40 ml) and water (4 ꢀ 40 ml), dried,
and concentrated under reduced pressure below 308C.
The residue was chromatographed on silica gel, eluting
with EtOAc/petroleum ether (4:1), and the eluant was
concentrated under reduced pressure below 308C to a
small volume and diluted with hexane to give 14
(1.44 g, 67%) as an unstable yellow oil. 1H NMR
[(CD3)2SO] d 8.92 (s, 1H), 8.76 (d, J ¼ 2:8 Hz, 1H), 8.34 (d,
J ¼ 2:8 Hz, 1H), 4.28 (t, J ¼ 5:4 Hz, 2H), 3.58 (t, J ¼
7:3 Hz, 2H), 3.51–3.43 (m, 4H), 3.13 (s, 3H), 1.43 (s, 18H).
A solution of 11 (2.70 g, 5.27 mmol) in TFA (12 ml) was
stirred at room temperature for 3 h, then diluted with
water (35 ml) to complete precipitation of the product.
Recrystallization from EtOAc/iPr2O gave 12 (2.19 g,
91%) as a yellow solid: m.p. 137–1388C. 1H NMR
[(CD3)2SO] d 14.18 (br, 1H), 8.82 (d, J ¼ 2:8 Hz, 1H),
8.60 (d, J ¼ 2:8 Hz, 1H), 4.27 (t, J ¼ 5:4 Hz, 2H),
3.59–3.46 (m, 6H), 3.12 (s, 3H). Analytically required
for C12H14BrN3O9S.0.5H2O: C, 30.98; H, 3.25; N,
9.03. Found: C, 32.12;32.17; H, 3.10;3.07; N,
9.18;9.23.
2-[(2-Bromoethyl)-2-[[(2-hydroxy[1,1,2,2-2H4]ethyl)-
amino]carbonyl]-4,6-dinitroanilino]ethyl methane-
sulfonate (13)
A suspension of powdered 12 (3.12 g, 6.84 mmol) in
CH2Cl2 (40 ml) was treated with oxalyl chloride
(0.88 ml, 10.3 mmol) and DMF (1 drop) and stirred at
room temperature for 1 h. Evaporation of the volatiles
under reduced pressure below 258C, followed by
azeotroping with benzene gave the crude acid
chloride. This was dissolved in THF (15 ml) and added
dropwise to a stirred solution of 2-amino[1,1,2,2-2-
H4]ethanol (0.58 g, 8.91 mmol) (Cambridge Isotope
Laboratories, Andover, MA 01810, USA) and Et3N
(1.43 ml, 10.3 mmol) in dioxane/THF (1:1) (25 ml)
at 08C. The mixture was stirred at 08C for a further
10 min, then poured into ice-cold 0.1 N aqueous
MsOH (120 ml) and extracted with EtOAc (2 ꢀ 80 ml).
The combined organic phase was washed with
water (2 ꢀ 40 ml), dried, and concentrated under re-
duced pressure. Chromatography on silica gel,
eluting with EtOAc, followed by precipitation of the
product from a CH2Cl2 solution with hexane, gave 13
(2.96 g, 86%) as a yellow gum; 1H NMR [(CD3)2SO]
d 8.74 (d, J ¼ 2:8 Hz, 1H), 8.71 (s, 1H), 8.35 (d, J ¼
2:8 Hz, 1H), 4.76 (s, 1H), 4.28 (t, J ¼ 5:5 Hz, 2H),
3.60–3.54 (m, 2H), 3.51–3.43 (m, 4H), 3.13 (s, 3H).
13C NMR [(CD3)2SO] d 165.3, 145.8, 145.3, 140.9,
136.2, 127.5, 122.1, 67.5, 58.4 (br, CD2), 5.43, 51.0,
41.3 (br CD2), 36.5, 29.7. HRMS(FAB) calculated, for
2-[(2-Bromoethyl)-2,4-dinitro-6-[[[2-(phosphonooxy)
[1,1,2,2-2H4]ethyl]-amino]carbonyl]anilino]ethyl
methanesufonate (1-D4)
A stirred solution of 14 (730 mg, 1.05 mmol) in CH2Cl2
(5 ml) was treated with TFA (5 ml) for 1 h at room
temperature, then concentrated to near dryness under
reduced pressure below 308C. The residual gum was
dissolved in dry MeCN (0.5 ml) and the solution was
diluted with dry CH2Cl2 (25 ml), filtered, and then
refrigerated at 58C for 30 h. The separated solid was
collected, washed with CH2Cl2, and dried under high
vacuum at room temperature to give 1-D4 (529 mg,
86%) as yellow crystals: m.p. 133–1358C. HPLC
analysis indicated the product was 94.7% pure, with
4.7% of the corresponding chloromesylate. 1H NMR
[(CD3)2SO] d 8.90 (s, 1H), 8.75 (d, J ¼ 2:8 Hz, 1H), 8.37
(d, J ¼ 2:8 Hz, 1H), 4.28 (t, J ¼ 5:4 Hz, 2H), 3.58
(t, J ¼ 7:0 Hz, 2H), 3.51–3.43 (m, 4H), 3.13 (s, 3H).
P(OH)2 signals not observed. 13C NMR [(CD3OD] d
168.4, 147.7, 147.6, 143.5, 137.8, 128.9, 123.8, 69.2,
65.0, (br, CD2), 56.9, 53.4, 41.0 (br, CD2), 37.3, 29.7.
HRMS(FAB) calculated for C14H17D749BrN4O12PS (MH+)
m/z 583.0049; found, 583.0050.
C
14H16D749BrN4O9S (MH+) m/z 503.0385; found,
Acknowledgements
503.0375.
HPLC analysis of the product indicated the presence
of 3.4% of the corresponding chloromesylate.
This work was supported by programme grant 01/276
from the Health Research Council of New Zealand, and
Copyright # 2007 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 7–12
DOI: 10.1002.jlcr

12 G. J. ATWELL AND W. A. DENNY
by Proacta Therapeutics Ltd. We thank Dianne Ferry
for conducting the specific activity measurements on
compound 1-T and Bill Wilson for helpful discussions.
GJ, Yang S, Denny WA, Wilson WR. Cancer Res,
submitted for publication.
3. Wilson WR, Pullen SM, Hogg A, Helsby NA,
Hicks KO, Denny WA. Cancer Res 2002; 62:
1425–1432.
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Copyright # 2007 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 7–12
DOI: 10.1002.jlcr