Development of novel quinone phosphorodiamidate prodrugs targeted to DT-diaphorase

Article abstract of DOI:10.1021/jm000179o

A series of naphthoquinone and benzimidazolequinone phosphorodiamidates has been synthesized and studied as potential cytotoxic prodrugs activated by DT-diaphorase. Reduction of the quinone moiety in the target compounds was expected to provide a pathway for expulsion of the phosphoramide mustard alkylating agent. All of the compounds synthesized were excellent substrates for purified human DT-diaphorase (k(cat)/K(m) = 3 x 10⁷ - 3 x 10⁸ M^-1 s^-1). The naphthoquinones were toxic to both HT-29 and BE human colon cancer cell lines in a clonogenic assay; however, cytotoxicity did not correlate with DT-diaphorase activity in these cell lines. The benzimidazolequinone analogues were 1-2 orders of magnitude less cytotoxic than the naphthoquinone analogues. Chemical reduction of the naphthoquinone led to rapid expulsion of the phosphorodiamidate anion; in contrast, the benzimidazole reduction product was stable. Michael addition of glutathione and other sulfur nucleophiles provides an alternate mechanism for activation of the naphthoquinone phosphorodiamidates, and this mechanism may contribute to the cytotoxicity of these compounds.

Full text of DOI:10.1021/jm000179o

J . Med. Chem. 2000, 43, 3157-3167
3157
Develop m en t of Novel Qu in on e P h osp h or od ia m id a te P r od r u gs Ta r geted to
DT-Dia p h or a se
Carolee Flader, J iwen Liu, and Richard F. Borch*
Departments of Chemistry and Pharmacology, University of Rochester, Rochester, New York 14642, and Department of
Medicinal Chemistry and Molecular Pharmacology and the Cancer Center, Purdue University, West Lafayette, Indiana 47907
Received April 24, 2000
A series of naphthoquinone and benzimidazolequinone phosphorodiamidates has been syn-
thesized and studied as potential cytotoxic prodrugs activated by DT-diaphorase. Reduction of
the quinone moiety in the target compounds was expected to provide a pathway for expulsion
of the phosphoramide mustard alkylating agent. All of the compounds synthesized were
excellent substrates for purified human DT-diaphorase (kcat/Km ) 3 × 107 - 3 × 108 M-1 s-1).
The naphthoquinones were toxic to both HT-29 and BE human colon cancer cell lines in a
clonogenic assay; however, cytotoxicity did not correlate with DT-diaphorase activity in these
cell lines. The benzimidazolequinone analogues were 1-2 orders of magnitude less cytotoxic
than the naphthoquinone analogues. Chemical reduction of the naphthoquinone led to rapid
expulsion of the phosphorodiamidate anion; in contrast, the benzimidazole reduction product
was stable. Michael addition of glutathione and other sulfur nucleophiles provides an alternate
mechanism for activation of the naphthoquinone phosphorodiamidates, and this mechanism
may contribute to the cytotoxicity of these compounds.
Sch em e 1
In tr od u ction
DT-diaphorase (E.C. 1.6.99.2, NAD(P)H:quinone-ac-
ceptor oxidoreductase) is a cytosolic FAD-containing
enzyme that catalyzes an obligatory two-electron reduc-
tion of a variety of quinones using NAD(P)H as cofactor.1
Although the physiological role of this enzyme is not
known, it is believed to involve the detoxification of
quinones by inhibiting formation of highly reactive
radical anion intermediates.2 Substantial evidence ex-
ists that DT-diaphorase is overexpressed in a number
of solid tumors,3 and the enzyme has become an attrac-
tive target for the design of bioreductively activated
prodrugs.4-6 The strategy underlying the selectivity of
these prodrugs is that two-electron reduction of the
quinone moiety markedly increases the electrophilicity
of appended functional groups, thereby converting the
prodrugs into DNA-reactive species. We have an ongo-
ing interest in the design of prodrugs that utilize
phosphoramidate release to deliver cytotoxins to tumor
cells.7 In this approach, intracellular reduction of the
prodrug generates an intermediate that should spon-
taneously release a cytotoxic phosphoramide mustard
that can cross-link DNA. We describe herein the syn-
thesis, activation mechanisms, and cytotoxicity of a
series of unique quinone phosphorodiamidates that are
excellent substrates for human DT-diaphorase and
exhibit significant cytotoxicity in vitro.
ates the corresponding hydroquinone in each case. The
naphthoquinone reduction product can expel the phos-
phorodiamidate anion directly. In the case of the benz-
imidazolequinone, reduction will increase electron den-
sity on the imidazole nitrogen and thereby facilitate
expulsion of the phosphorodiamidate. A series of naph-
thoquinone and benzimidazolequinone prodrugs con-
taining either N,N-bis(haloethyl)- or N,N′-haloethylphos-
phorodiamidates were prepared (Chart 1). In addition,
compounds containing a novel bis[N-methyl-N-(2-halo-
ethyl)]phosphorodiamidate (6a -c) were synthesized. It
was hypothesized that this bis-alkylating moiety might
be more cytotoxic than compounds containing an N,N-
bis(2-haloethyl)amine substituent because of the pre-
sumed faster rate of aziridinium ion formation resulting
Resu lts a n d Discu ssion
Ch em istr y. The mechanistic rationale for the pro-
posed activation of these compounds is outlined in
Scheme 1. Two-electron reduction of the quinone gener-
* Address for correspondence: Department of Medicinal Chemistry
and Molecular Pharmacology, Purdue University, West Lafayette, IN
47907. Tel: (765) 494-1403. Fax: (765) 494-1414. E-mail: rickb@
pharmacy.purdue.edu.
10.1021/jm000179o CCC: $19.00 © 2000 American Chemical Society
Published on Web 07/18/2000

3158 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 16
Flader et al.
N-methyl-N-(2-bromoethyl)amine hydrobromide.10 Bis-
(2-bromoethyl)amine hydrobromide (7)11 was phospho-
rylated in a similar manner, except that the reaction
temperature was maintained below 0 °C. Surprisingly,
at higher temperatures a side product [N-(2-chloroet-
hyl)-N-(2-bromoethyl)phosphoramidic dichloride] was
formed that could not be separated from the desired
product. The side product presumably arises from the
displacement by chloride released in the phosphoryla-
tion reaction. Compounds 12 and 13a -f were generated
by reaction of alcohols 11a -d with the respective
phosphoramidic dichloride followed by reaction with
ammonia. 1,4-Dimethoxynaphthalenes 12 and 13a -e
were oxidized to their respective quinones 2 and 3a -e
using ceric ammonium nitrate,12 and quinone 3f was
generated from 13f using silver(II) oxide as oxidant.13
The acetate 16b,14 was prepared by acetylation of alcohol
11a followed by oxidation with ceric ammonium nitrate.
Isophosphoramide mustard prodrug 5 was synthesized
in two steps from alcohol 11a , phosphorus oxychloride,
and N-(2-chloroethyl)amine hydrochloride.
Ch a r t 1
from the inductive effect of the N-methyl substituent.
Finally, two compounds (acetate 1 and morpholine
phosphorodiamidate 2) that do not contain an alkylating
moiety were prepared. They were used to assess the
substituent effect on enzyme substrate activity and the
contribution of the quinone methide product to the
cytotoxicity of the prodrugs.
The synthesis of the target naphthoquinone and
benzimidazolequinone phosphorodiamidates was carried
out as shown in Schemes 2-4. Naphthoquinones 1, 2,
3a-f, 5, and 6a-c were synthesized from 1,4-dimethoxy-
2-(hydroxymethyl)naphthalene (11a ), which was pre-
pared by complete methylation8 of 1,4-dihydroxy-2-
naphthoic acid followed by reduction of the ester to the
alcohol (Scheme 2). Substituted alcohols 11b-d were
prepared by deprotonation of 11a with n-butyllithium
followed by reaction with electrophiles methyl iodide,
dimethyl disulfide, and 1,2-dibromotetrafluoroethane,9
respectively. Phosphorylating agent 9 was prepared
according to the procedure used to phosphorylate
Compounds 4a ,b were synthesized by nitration of 1,4-
dimethoxybenzene to give a 8:1 ratio of 2,3- and 2,5-
dinitro-1,4-dimethoxybenzenes (Scheme 3).15 The mix-
ture was reduced to the diamines16 with stannous
chloride and reacted with glycolic acid17 to give benz-
imidazole 17. Compound 17 was methylated and the
product (18) phosphorylated as described above to give
19a ,b, which were converted to the benzimidazole-
quinones 4a ,b by silver(II) oxide oxidation.
Naphthoquinone prodrugs containing a bis[N-methyl-
N-(2-bromoethyl)]- (6a ,b) or bis[N-methyl-N-(2-chloro-
ethyl)]phosphorodiamidate (6c) were prepared using
phosphorus(III) chemistry as outlined in Scheme 4.
Alcohol 11a or 11b was reacted with phosphorus
trichloride in the presence of base at -70 °C. Two
equivalents of the N-methyl-N-(2-haloethyl)amine hy-
drohalide10 were then added, and without isolation, the
intermediate was oxidized with tert-butyl hydroperox-
Sch em e 2a
a
Reagents: (a) NEt3, CH2Cl2, -40 to 0 °C; (b) NEt3, CH2Cl2, 0 °C to rt; (c) CH3I, K2CO3, acetone, reflux; (d) LiAlH4, ether, reflux; (e)
nBuLi, THF, -78 °C to rt, then CH3I, CH3SSCH3 or F2BrCCBrF2; (f) LiN(TMS)2, THF, -78 °C, then 8, 9, or Cl2P(O)N(CH2CH2Cl)2, -78
°C, then NH3, -20 °C; (g) Ce(NH4)2(NO3)6, CH3CN/H2O; (h) AgO, THF, 6 N HNO3; (i) CH3COCl, NEt3, CH2Cl2; (j) iPr2NEt, CH2Cl2, -78
°C to rt, then 11a + nBuLi, -78 °C to rt.

Quinone Phosphorodiamidate Prodrugs
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 16 3159
Sch em e 3a
Ta ble 1. Kinetic Analysis of Naphthoquinones and
Benzimidazolequinonesa
Km
(µM)
Vmax
(µM/s)
kcat
(1/s)
kcat/Km
compound
Vmax/Km
(M-1 s-1
)
menadioneb
1.2
1.7
0.60b
2.4
0.50b
1.4
0.83
2.0
0.81
1.8
0.76
1.1
700
280
15
46
63
51
78
72
97
66
41
88
115
180
190
5.8 × 108
1.6 × 108
1.6 × 107
2.2 × 108
9.1 × 107
2.0 × 108
8.6 × 107
1.2 × 108
3.0 × 108
3.1 × 107
4.5 × 107
2.3 × 108
1.3 × 108
5.0 × 107
1.4 × 108
1
2
0.98
0.21
0.69
0.26
0.91
0.59
0.32
2.1
0.91
0.38
0.89
3.6
0.81
0.41
0.56
0.46
0.69
0.64
0.86
0.59
0.37
0.78
1.0
3a
3b
3c
3d
3e
3f
4a
4b
5
2.7
0.28
0.40
2.1
1.1
0.44
1.2
6a
6b
6c
1.6
1.7
a
Reagents: (a) HNO3, 90 °C; (b) SnCl2, HCl, 100 °C, then
1.4
glycolic acid, H2O, reflux; (c) LiN(TMS)2, 0 °C to rt, then CH3I; (d)
LiN(TMS)2, THF, -78 °C, then 8 or Cl2P(O)N(CH2CH2Cl)2, -78
°C, then NH3, -20 °C; (e) AgO, THF, 6 N HNO3.
a
See Experimental Section for details of enzyme kinetics assays.
b
Enzyme assays with menadione were conducted using 1/10 of
the enzyme concentration used for the other assays.
Sch em e 4a
Ta ble 2. Clonogenic Survival of HT-29 and BE Cellsa
b
b
LC99 (µM)
LC99 (µM)
HT-29 BE
4.1 3.3
130 120
300
7.8
compound
HT-29
BE
compound
1
2
64
89
22
72
3f
4a
4b
5
6a
6b
6c
3a
3b
3c
3d
3e
7.8
1.7
11
4.6
1.2
13
220
14
4.5
1.2
2.1
4.2
1.0
5.9
1.5
4.5
0.44
2.8
a
Reagents: (a) PCl3, iPr2NEt, CH2Cl2/CH3CN, -70 °C, then 2
equiv HX-HN(CH3)CH2CH2X, iPr2NEt, -60 to -50 °C, then
tBuOOH, -40 to -20 °C; (b) Ce(NH4)2(NO3)6, CH3CN/H2O.
a
2-h drug treatment; see Experimental Section for details of
the clonogenic assay. Concentration that reduces clonogenic
b
survival by 2 log units (99%).
ide. The resulting dimethoxynaphthalene phosphoro-
diamidates 20a -c were oxidized to their respective
naphthoquinones 6a -c using ceric ammonium nitrate.
En zym e Kin etics. Each of the quinones was assayed
spectrophotometrically at 25 °C with purified human
DT-diaphorase to determine its enzyme kinetic param-
eters. A standard coupled assay monitoring the absor-
bance change of cytochrome c was used.18 All of the
compounds exhibited some degree of product and sub-
strate inhibition at substrate concentrations near KM,
of the 2-h drug treatments are summarized in Table 2.
Several important structure-activity relationships are
apparent from these data. First, the presence of an
incipient alkylating group markedly enhances cytotox-
icity; the cytotoxicity of the nonalkylating morpholine
phosphorodiamidate 2 is approximately 1 order of
magnitude less than that of the alkylating phosphoro-
diamidates. Second, introduction of the 3-methyl sub-
stituent enhances the cytotoxicity of the naphthoquino-
nes in both cell lines. Third, cytotoxicity is not strongly
dependent on the nature of the alkylating group; while
the bis(methyl bromoethyl)phosporodiamidate com-
pounds are somewhat more cytotoxic, the isophosphor-
amide prodrug 5 is comparable in cytotoxicity to the
bis(haloethyl) compounds.
Two unexpected results emerged from the cytotoxicity
experiments. First, the benzimidazolequinone analogues
4a ,b are essentially inactive in this assay. The enzyme
kinetics experiments suggest that these compounds are
excellent substrates for the enzyme, indicating that
there may be a problem with the phosphorodiamidate
expulsion step. Second, cytotoxicity is comparable in the
HT-29 and BE cell lines. It was expected that the
quinone phosphorodiamidates would be significantly
more toxic to the HT-29 cells because of the high level
of enzyme activity, but this was not the case. Compound
3a was evaluated in the NCI human tumor cell line in
vitro screen. The compound showed modest cytotoxicity
across all cell lines but showed significantly greater
cytotoxicity against melanoma (ca. 1 order of magnitude
more potent than the mean for 5/8 cell lines). Examina-
tion of the GI50 values for 3a and cellular levels of DT-
so a Hanes analysis was used to determine KM and Vmax
.
Reaction velocities were obtained from the initial, linear
portion of the reaction progress curve. The results of
the kinetic analyses are shown in Table 1; menadione
is included for comparison. All of the haloethylphospho-
rodiamidates are excellent substrates for the enzyme
(kcat/KM > 3 × 107 M-1 s-1), and the differences in KM
and Vmax among the haloethylphosphorodiamidates are
modest. Introduction of a 3-methyl substituent in the
naphthoquinone results in a 3-4-fold increase in KM (3a
vs 3b, 3c vs 3d , 6a vs 6b). Bis(methyl haloethyl)
compounds 6a -c had somewhat higher KM and Vmax
values than the other haloethylphosphorodiamidates,
and the benzimidazolequinones had slightly lower turn-
over numbers than the other compounds.
In Vitr o Cytotoxicity. The cytotoxicity of these
prodrugs was assessed using HT-29 and BE human
colon carcinoma cell lines. HT-29 cells have a high level
of DT-diaphorase activity (1410 nmol cytochrome c
reduced/min/mg protein). In contrast, the BE cell line
has a C-to-T point mutation in the gene that encodes
for DT-diaphorase, so this cell line produces a mutant
that has no measurable enzymatic activity.19 The results

3160 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 16
Flader et al.
solvolysis products within 3 min at room temperature.
The half-lives of the phosphorodiamidates resulting
from reduction of 3a (t1/2 ) 12 min at room temperature)
and 6c (t1/2 ) 2.9 min at 37 °C) were determined by
linear regression analysis of the disappearance of the
phosphorodiamidate resonance. Two N-methyl-N-(2-
bromoethyl)amine substituents on phosphorus (phos-
phorodiamidate from 6a , t1/2 < 1 min) increase the rate
of aziridinium ion formation by at least 1 order of
magnitude when compared to the N,N-bis(2-bromoeth-
yl)phosphorodiamidate from 3a . Similarly, cyclization
of the bis[N-methyl-N-(2-chloroethyl)]phosphorodiami-
date (from 6c) at 37 °C is a factor of 8 faster than the
N,N-bis(2-chloroethyl) analogue (from 3c, t1/2 ) 23 min)
and a factor of 22 faster than the N,N′-bis(2-chloroethyl)
analogue (from 5, t1/2 ) 64 min). These differences
presumably result from the electron-donating effects of
the N-methyl group compared to the electron-withdraw-
ing effects of a second haloethyl group.
F igu r e 1. Reduction of quinone phosphorodiamidate 3c with
sodium dithionite (3 equiv, 3:4 CH3CN:0.4 M cacodylate buffer,
pH ∼ 7.4, 37 °C): A, phosphoramide mustard; B, monosub-
stituted product; C, disubstituted product; (i) 0, (ii) 5, (iii) 13,
(iv) 21, and (v) 29 min. Chemical shifts are reported relative
to the TPPO reference. See Results and Discussion for details.
The chemical reduction of benzimidazolequinone phos-
phorodiamidate 4a was also carried out using sodium
dithionite as described above. In contrast to the naph-
thoquinones, however, the resulting hydroquinone was
stable for >12 h and did not expel the phosphorodia-
midate anion. This result provides an explanation for
the absence of cytotoxicity for 4a ,b; although the pro-
drugs are good substrates for DT-diaphorase, the phos-
phorodiamidate anion is not appreciably released from
the resulting hydroquinone.
diaphorase20 showed that a weak correlation exists
(Pearson correlation coefficient ) 0.31) between growth
inhibition and DT-diaphorase activity. Presumably ac-
tivation mechanisms other than quinone reduction must
be contributing to the cytotoxicity of these compounds.
Ch em ica l Activa tion of P h osp h or od ia m id a tes.
The cytotoxicity of the quinone phosphorodiamidates
presumably relies on the facile release of the phospho-
rodiamidate anion following reduction to the hydro-
quinone. If the phosphorodiamidate anion is released
too slowly, the hydroquinone can diffuse out of the cell
in which it was activated and the selective delivery of
the active drug will be decreased. Reoxidation of the
hydroquinone by oxygen can also occur if the release
step is too slow. It is therefore important to ascertain
whether these prodrugs deliver the alkylating moiety
quickly and efficiently. To this end, the chemical reduc-
tion of the naphthoquinone prodrug 3c using sodium
dithionite (3 equiv, 3:4 CH3CN:0.4 M cacodylate buffer,
pH ∼ 7.4, 37 °C) was followed using 31P NMR; the
results are shown in Figure 1. The resonance for
quinone 3c at -5.3 ppm (spectrum i) had disappeared
within 5 min and was replaced by the resonance for
phosphoramide mustard A at -12.2 ppm (spectrum ii).
Similar results were obtained for compounds 3a , 5, and
6a . The subsequent alkykation chemistry previously
reported by Ludeman et al.21 was also observed, except
that in this case the mono- (B) and bis- (C) bisulfite
substitution products (-12.0 and -11.8 ppm, respec-
tively; spectra iii and iv) were formed instead of glu-
tathione products. Bisulfite, a product of the dithionite
reduction and a contaminant in commercial sodium
dithionite, is a stronger nucleophile than water so that
attack of bisulfite on the aziridinium ion intermediate
predominates over alkylation and hydrolysis reactions.22
The chemical reductions of naphthoquinones 3a and
6a ,c were also monitored by 31P NMR in order to
estimate the half-lives for the cyclization of the resulting
phosphorodiamidate anions. The phosphoramidate ob-
tained from reduction of 6a was the most reactive; 80%
of the phosphorodiamidate anion was converted to
Oth er Cellu la r Mech a n ism s of Qu in on e Activa -
tion . The absence of a correlation between DT-diapho-
rase activity and in vitro cytotoxicity of the quinone
phosphorodiamidates suggests that the cells are using
other mechanisms to activate the compounds, and an
attempt was made to determine what those mechanisms
might be. Other enzymes that might be capable of
activating the compounds in HT-29 and BE cells were
considered; the levels of cytochrome b5 reductase and
cytochrome P-450 reductase in the cell lines20 were
investigated. The activities of these enzymes (40-65
nmol cytochrome c reduced/min/mg protein) were nearly
equivalent for the two cell lines and were much lower
than the DT-diaphorase levels found in HT-29 cells.
Furthermore, no correlation was observed between the
GI50 values of 3a and the levels of these enzymes in the
NCI panel.
The Michael addition of sulfur nucleophiles to naph-
thoquinones is well-known,23 and addition to the 3-posi-
tion might provide a pathway for phosphorodiamidate
anion release. Two possible products could be predicted
from the Michael addition of a nucleophile to the
naphthoquinone (Scheme 5): addition at the 2-position
(route b) leads to reversible formation of the kinetic
product;23 addition at the 3-position (route a) would give
an intermediate that could expel the nucleophile in the
reversible reaction or could expel the phosphorodiami-
date anion in an irreversible step. Preliminary experi-
ments were carried out using sodium dimethyldithio-
carbamate (DDTC), which contains a highly nucleophilic
sulfur that is anionic at physiologic pH. Activation of
3a with DDTC (3 equiv, 3:4 THF:0.4 M cacodylate
buffer, pH ∼ 7.3, room temperature) was monitored
using 31P NMR. Three equivalents of nucleophile were
used, assuming that one equivalent would be consumed

Quinone Phosphorodiamidate Prodrugs
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 16 3161
Sch em e 5
To assess whether the intramolecular glutathione
reaction might be affecting the activity of 3a in vitro,
the NCI data were examined to determine whether
growth inhibition correlates with glutathione S-trans-
ferase activity (GST-P or GST-M) or with cellular
glutathione levels.25 No correlation was observed with
GST-P or GST-M activities; however, a correlation was
observed between GI50 and glutathione levels (Pearson
correlation coefficient ) 0.43). This result suggests that
intracellular glutathione may be contributing to the
intracellular activation of the naphthoquinones.
In Vivo Cytotoxicity. The antitumor activity of 3a
was evaluated in a xenograft model. Nude mice were
injected subcutaneously with human renal carcinoma
cells (A4982LM), and the tumors were allowed to grow
until a measurable mass was present (22 days). The
A4982LM cell line is a highly metastatic subline of A498
with high levels of DT-diaphorase (1010 nmol cyto-
chrome c reduced/min/mg protein). A single dose of 3a
(25 mg/kg) was administered intraperitoneally, and the
tumor size was monitored for 7 weeks. Compound 3a
modestly retarded tumor growth, exhibiting a 40%
reduction in tumor size at all time points.
by the Michael addition and the other two would be
consumed by reaction with the resulting phosphorodi-
amidate anion. The resonance for quinone 3a (-5.7
ppm) had disappeared and was replaced by the reso-
nance for the phosphorodiamidate anion at -13.1 ppm
within 5 min after addition of DDTC, confirming that
nucleophilic activation of the naphthoquinone is rapid
and complete. Subsequent phosphorodiamidate anion
chemistry was also observed, generating primarily the
mono- and bis-DDTC substitution products at -12.9 and
-12.6 ppm, respectively. Reaction of 3a with one
equivalent of DDTC resulted in complete conversion to
the phosphorodiamidate anion within 3.5 min after
addition of DDTC, suggesting that nucleophilic activa-
tion of 3a is complete before cyclization of the phospho-
rodiamidate anion to the aziridinium ion takes place.
The experiments described above show that com-
pound 3a is activated by a potent sulfur nucleophile. It
was of interest to know whether glutathione might react
in the same way. Glutathione, the primary nonprotein
intracellular thiol, is responsible for maintaining the
cellular redox environment and removing potential
electrophilic cytotoxins. It is overproduced (1-10 mM
intracellular concentrations) in many cancer cell lines,
particularly those that have acquired resistance to
anticancer drugs.24,25 Thus the reaction of 3c with
glutathione (3 equiv, 1:1.3 CH3CN:0.4 M cacodylate
buffer, pH ∼ 7.5, room temperature) was monitored by
31P NMR. The reaction with glutathione was essentially
identical to that of DDTC; the resonance for quinone
3c had disappeared and was replaced by the resonance
for phosphorodiamidate anion within 4 min of glu-
tathione addition. The result was essentially identical
when only one equivalent of glutathione was used, again
suggesting that activation of this compound is complete
before cyclization of the phosphorodiamidate anion can
occur.
Con clu sion
A series of naphthoquinone and benzimidazolequino-
ne phosphorodiamidates has been prepared in the
expectation that they would function as mechanism-
based anticancer drugs. All of the compounds are
excellent substrates for DT-diaphorase. The naphtho-
quinones undergo facile activation and expulsion of the
cytotoxic phosphorodiamidate either via reduction or
reaction with glutathione. The naphthoquinone ana-
logues are potent cytotoxic agents, but they do not
exhibit selectivity in vitro for cells with high levels of
DT-diaphorase, presumably because of the glutathione-
catalyzed activation in cells with high glutathione levels.
Phosphorodiamidate anions containing the bis(N-meth-
yl-N-haloethyl) moiety cyclize to the aziridinium ion
approximately 1 order of magnitude faster than those
containing the bis(haloethyl)phosphorodiamidate, but
this increase in reactivity results in only a modest
increase in cytotoxicity. The benzimidazolequinone phos-
phorodiamidates were not cytotoxic in vitro, because the
hydroquinone produced by enzymatic activation does not
eliminate the cytotoxic phosphorodiamidate anion.
Exp er im en ta l Section
Gen er a l Meth od s. All 1H NMR spectra were measured on
a 250-MHz Bruker NMR system equipped with a multinuclear
(1H, 13C, 19F and 31P) 5-mm probe. The NMR data acquisition/
processing program MacNMR was used with the Tecmag data
acquisition system. 1H chemical shifts are reported in parts
per million from tetramethylsilane. All 31P NMR spectra were
obtained on the same instrument using broadband gated
decoupling and a pulse width of 10 µs. Chemical shifts are
reported in parts per million from a coaxial insert containing
5% phosphoric acid in H2O (structure characterization) or 1%
triphenylphosphine oxide (TPPO) in benzene-d6 (31P kinetics);
the chemical shift difference between these references is 24.7
ppm. All variable temperature experiments were conducted
using a Bruker variable temperature unit. Chromatographic
purifications were carried out by flash chromatography using
silica gel grade 60. Melting points were determined on a Mel-
Temp II apparatus and are uncorrected. Elemental analyses
were performed by the Purdue University Microanalysis Lab,
West Lafayette, IN. Mass spectral data were obtained from
A study of the activation of the 3-substituted naph-
thoquinones by glutathione was then undertaken. It was
hypothesized that both the steric and electronic effects
of the substituents might influence the rate of glu-
tathione activation. The reactions of the naphtho-
quinone phosphorodiamidates substituted with a methyl
(3d ), 3-methylthio (3e), or bromo (3f) group were carried
out using three equivalents of glutathione. In each case
the quinone was completely converted to its respective
phosphorodiamidate anion within 4 min at room tem-
perature, suggesting that substituent effects are mini-
mal in this reaction.

3162 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 16
Flader et al.
the Purdue University Mass Spectrometry Service, West
Lafayette, IN, using fast atom bombardment with a 3-ni-
trobenzyl alcohol matrix. A glass calomel electrode on either
a radiometer pH meter or an Orion PerpHect LogR meter,
model 330, was used for acidity measurements. IR spectra
were recorded on a Nicolet Magna IR 550 spectrometer using
either a thin film or Nujol suspension, as noted, between NaCl
plates. All anhydrous reactions were carried out in either a
flame-dried or oven-dried flask under argon. All organic
solvents were distilled prior to use.
Bis(2-br om oeth yl)a m in e Hyd r obr om id e (7). According
to a modification of the procedure described by Cortese,11
hydrobromic acid (75 mL, 0.45 mol, 48% by wt) was added
slowly, with stirring, to diethanolamine (11.0 g, 0.10 mol) at
0 °C. The reaction mixture was heated to reflux and then water
was removed by distillation through a vigreaux column. The
distillate temperature was 100 °C for the first 15 mL collected
and 125 °C for the remainder. After a total of 40 mL of
distillate was collected, additional 48% HBr (50 mL, 0.30 mol)
was added and 50 mL of distillate was collected. Hydrobromic
acid (25 mL, 48%) was added and the reaction mixture was
refluxed overnight. The reaction mixture was then distilled
(∼30 mL collected) and the still pot residue was poured into
acetone at -78 °C. The white solid that precipitated (10.3 g,
22% diethanolamine hydrobromide and 78% product 7 as
determined by 1H NMR) was collected by filtration. This
impure product can either be used directly in the subsequent
phosphorylation reaction or can be purified by repeated
recrystallization from acetone. This recrystallization procedure
actually crystallizes out diethanolamine hydrobromide, so that
purer product 7 is recovered from the filtrate upon successive
recrystallizations: 1H NMR (D2O) δ 3.55 (t, 4H), 3.45 (t, 4H);
1H NMR of diethanolamine hydrobromide (D2O) δ 3.78 (t, 4H),
3.12 (t, 4H).
1,4-Dim eth oxy-2-h yd r oxym eth yln a p h th a len e (11a ). A
solution of lithium aluminum hydride (29.2 mL, 29.2 mmol, 1
M in ether) in ether (30 mL) was heated to reflux under argon.
A solution of 10 (7.18 g, 29.2 mmol) in ether (30 mL) and THF
(8 mL) was added dropwise over 35 min. The resulting mixture
was refluxed for 3 h and then cooled to 0 °C. Methanol (10
mL) was added dropwise and the resulting clear yellow
solution was stirred for 1 h. Saturated NH4Cl (20 mL) and
aqueous HCl (5 mL, 10%) were added and the mixture was
extracted with ether (6×). The combined organic layers were
washed with saturated Na2CO3 (2×) and H2O (2×), dried
(MgSO4), filtered and evaporated. Column chromatography of
the crude product (35:65 EtOAc:hexanes) afforded 11a (5.82
g, 92%) as a pink solid: Rf 0.48 (35:65 EtOAc:hexanes); mp
69-70 °C; 1H NMR (CDCl3) δ 8.23 (dd, 1H), 8.03 (dd, 1H), 7.52
(m, 2H), 6.82 (s, 1H), 4.89 (s, 2H), 3.99 (s, 3H), 3.92 (s, 3H),
1.95 (bs, 1H).
1,4-Dim et h oxy-3-m et h yl-2-h yd r oxym et h yln a p h t h a -
len e (11b). n-Butyllithium (7.3 mL, 18.3 mmol, 2.5 M in
hexanes) was added dropwise via syringe to a solution of 11a
(1.00 g, 4.58 mmol) in THF (30 mL) at -78 °C under argon.
The solution was slowly warmed to room temperature and
stirred for 1 h. Methyl iodide (0.34 mL, 5.50 mmol) was added
dropwise and the solution was stirred for 20 min. Water (8
mL) was added and the mixture was extracted with EtOAc
(4×). The combined organic layers were dried (MgSO4), filtered
and evaporated. Column chromatography of the crude product
(45:55 EtOAc:hexanes) afforded 11b (0.61 g, 57%) as a yellow
solid: Rf 0.59 (45:55 EtOAc:hexanes); mp 116-117 °C; 1H
NMR (CDCl3) δ 8.07 (dd, 2H), 7.51 (m, 2H), 4.94 (s, 2H), 3.98
(s, 3H), 3.88 (s, 3H), 2.52 (s, 3H), 1.73 (bs, 1H).
1,4-Dim et h oxy-3-t h iom et h yl-2-h yd r oxym et h yln a p h -
th a len e (11c). n-Butyllithium (0.73 mL, 1.83 mmol, 2.5 M in
hexanes) was added dropwise to a solution of 11a (100 mg,
0.458 mmol) in THF (6 mL) at -78 °C under argon. The
solution was slowly warmed to room temperature and stirred
for 1 h. Dimethyl disulfide (0.050 mL, 0.55 mmol) was added
dropwise and the resulting mixture was stirred for 30 min.
Water (1 mL) was added and the mixture was extracted with
EtOAc (3×). The combined organic layers were washed with
saturated NaCl, dried (MgSO4), filtered and evaporated.
Column chromatography of the crude product (20:80 EtOAc:
hexanes) afforded 11c (71.2 mg, 59%, 82% based on recovered
11a ) as an oil: Rf 0.26 (20:80 EtOAc:hexanes); 1H NMR
(CDCl3) δ 8.10 (m, 2H), 7.55 (m, 2H), 5.07 (s, 2H), 4.03 (s, 3H),
3.99 (s, 3H), 2.53 (s, 3H), 1.60 (bs, 1H).
1,4-Dim et h oxy-3-b r om o-2-h yd r oxym et h yln a p h t h a -
len e (11d ). n-Butyllithium (2.9 mL, 7.33 mmol, 2.5 M in
hexanes) was added dropwise to a solution of 11a (400 mg,
1.83 mmol) in THF (25 mL) at -78 °C under argon. The
reaction mixture was slowly warmed to room temperature and
stirred for 1 h. 1,2-dibromotetrafluoroethane (0.26 mL, 2.20
mmol) was added dropwise and the solution was stirred for 1
h. Water (10 mL) was added and the mixture was extracted
with EtOAc (3×). The combined organic layers were dried
(MgSO4), filtered and evaporated. Column chromatography of
the crude product (35:65 EtOAc:hexanes) afforded 11d (273
mg, 50%, 63% based on recovered 11a ) as a yellow solid: Rf
0.62 (35:65 EtOAc:hexanes); mp 115-117 °C; 1H NMR (CDCl3)
δ 8.11 (m, 2H), 7.57 (m, 2H), 5.03 (s, 2H), 4.02 (s, 3H), 3.99 (s,
3H), 2.36 (bs, 1H).
Bis(2-br om oeth yl)ph osph or am idic Dich lor ide (8). Phos-
phorus oxychloride (0.30 mL, 3.2 mmol) was added slowly to
a suspension of 7 (1.00 g, 3.2 mmol) in CH2Cl2 (22 mL) at -40
°C under argon. Triethylamine (1.20 mL, 8.6 mmol) was added
dropwise via syringe over 5 min with vigorous stirring to avoid
local heating. The cloudy white reaction mixture was warmed
to 0 °C over 2.5 h and stirred at 0 °C for 4 h. Saturated
ammonium chloride (8 mL) was added and the mixture was
extracted with CH2Cl2 (3×). The combined organic layers were
dried (MgSO4), filtered and evaporated. Column chromatog-
raphy of the crude product (20:80 EtOAc:hexanes) afforded 8
(0.76 g, 68%) as a white solid: Rf 0.56 (20:80 EtOAc:hexanes);
1
mp 43-44 °C; H NMR (CDCl3) δ 3.73 (dt, 4H), 3.55 (t, 4H);
31P NMR (CDCl3) δ 16.48.
Mor p h olin op h osp h or a m id ic Dich lor id e (9). Morpholine
(2.6 mL, 30 mmol) was added dropwise over 10 min to a
vigorously stirred solution of phosphorus oxychloride (2.8 mL,
30 mmol) in CH2Cl2 (25 mL) at 0 °C under argon. Triethy-
lamine (4.6 mL, 33 mmol) was added dropwise via syringe and
the resulting thick suspension was warmed to room temper-
ature and stirred overnight. The mixture was poured over ice
and extracted with CH2Cl2 (3×). The combined organic layers
were washed with saturated NaCl, dried (MgSO4), filtered and
evaporated. Column chromatography of the crude product (25:
75 EtOAc:hexanes) afforded 9 (4.27 g, 70%) as a pale yellow
liquid: Rf 0.41 (25:75 EtOAc:hexanes); 1H NMR (CDCl3) δ 3.75
(t, 4H), 3.38 (dt, 4H); 31P NMR (CDCl3) δ 14.70.
Meth yl 1,4-Dim eth oxy-2-n a p h th oa te (10). According to
a modification of the procedure of Brimble et al.,8 potassium
carbonate (30.50 g, 0.22 mol) and methyl iodide (27.5 mL, 0.44
mol) were added to a solution of 1,4-dihydroxy-2-naphthoic acid
(6.00 g, 0.029 mol) in acetone (120 mL) under argon. The
mixture was refluxed for 48 h. Water (50 mL) was added and
the mixture was extracted with CH2Cl2 (5×). The combined
organic layers were washed with H2O (2×), dried (MgSO4),
filtered and evaporated. Column chromatography of the crude
product (15:85 EtOAc:hexanes) afforded 10 (7.05 g, 97%) as a
solid: Rf 0.44 (15:85 EtOAc:hexanes); mp 48-50 °C; 1H NMR
(CDCl3) δ 8.24 (m, 2H), 7.59 (m, 2H), 7.16 (s, 1H), 4.02 (s, 3H),
4.01 (s, 3H), 4.00 (s, 3H).
2-(1,4-Dim et h oxyn a p h t h yl)m et h yl Mor p h olin op h os-
p h or od ia m id a te (12). Lithium bis(trimethylsilyl)amide (3.8
mL, 3.8 mmol, 1 M in THF) was added dropwise to a solution
of 11a (750 mg, 3.44 mmol) in THF (10 mL) at -78 °C under
argon. The resulting solution was stirred for 5 min and added
dropwise via syringe to a solution of 9 (840 mg, 4.13 mmol) in
THF (20 mL) at -78 °C. The reaction mixture was stirred at
-78 °C for 1.5 h and then warmed to -20 °C. Gaseous
ammonia was passed through the reaction mixture for 15 min.
Hydrochloric acid (2%, 40 mL) was added and the mixture was
extracted with EtOAc (6×). The combined organic layers were
washed with saturated NaCl (2×), dried (MgSO4), filtered and
evaporated. Column chromatography of the crude product (5:

Quinone Phosphorodiamidate Prodrugs
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 16 3163
95 MeOH:CHCl3) afforded 12 (960 mg, 76%) as a pale yellow
foam: Rf 0.13 (5:95 MeOH:CHCl3); 1H NMR (CDCl3) δ 8.24
(dd, 1H), 8.06 (dd, 1H), 7.54 (m, 2H), 6.85 (s, 1H), 5.23 (dd,
2H, J P,H ) 7.8 Hz), 4.00 (s, 3H), 3.94 (s, 3H), 3.63 (t, 4H), 3.19
(dt, 4H), 2.64 (bs, 2H); 31P NMR (CDCl3) δ 15.10.
in THF (10 mL) at -78 °C under argon. The resulting solution
was stirred for 5 min and added dropwise via syringe to a
solution of bis(2-chloroethyl)phosphoramidic dichloride (710
mg, 2.75 mmol) in THF (20 mL) at -78 °C. The reaction
mixture was stirred at -78 °C for 1.5 h and then was warmed
to -20 °C. Gaseous ammonia was passed through the reaction
mixture for 10 min. The mixture was stirred for an additional
10 min, aqueous HCl (2%, 30 mL) was added and the mixture
was extracted with EtOAc (4×). The combined organic layers
were washed with saturated NaCl (2×), dried (MgSO4), filtered
and evaporated. Column chromatography of the crude product
(2:98 MeOH:EtOAc) afforded 13c (480 mg, 50%) as a yellow
oil: Rf 0.59 (2:98 MeOH:EtOAc); 1H NMR (CDCl3) δ 8.25 (dd,
1H), 8.06 (dd, 1H), 7.54 (m, 2H), 6.85 (s, 1H), 5.33 (m, 2H,
J P,H ) 7.8 Hz), 4.01 (s, 3H), 3.94 (s, 3H), 3.65 (t, 4H), 3.48 (m,
4H), 2.77 (bs, 2H); 31P NMR (CDCl3) δ 15.72.
2-(3-Meth yl-1,4-d im eth oxyn a p h th yl)m eth yl N,N-bis(2-
ch lor oeth yl)p h osp h or od ia m id a te (13d ) was prepared from
11b as described for the synthesis of 13c (2.15-mmol scale).
Column chromatography of the crude product (1:99 MeOH:
EtOAc) afforded 13d (440 mg, 47%) as a pale yellow foam: Rf
0.44 (1:99 MeOH:EtOAc); 1H NMR (CDCl3) δ 8.08 (m, 2H), 7.53
(m, 2H), 5.31 (m, 2H, J P,H ) 6.8 Hz), 3.98 (s, 3H), 3.89 (s, 3H),
3.64 (t, 4H), 3.45 (dt, 4H), 2.77 (bs, 2H), 2.52 (s, 3H); 31P NMR
(CDCl3) δ 15.65.
2-(1,4-Na p h th oqu in on yl)m eth yl Mor p h olin op h osp h o-
r od ia m id a te (2). Ceric ammonium nitrate (1.03 g, 1.88 mmol)
in H2O (6 mL) was added in portions over 15 min to a solution
of 12 (300 mg, 0.82 mmol) in CH3CN (15 mL) at -8 °C. The
solution was stirred at -8 °C for 40 min and then was warmed
to 0 °C and stirred for 50 min. The solution was extracted with
CHCl3 (3×), and the combined organic layers were dried
(MgSO4), filtered and evaporated. Column chromatography of
the crude product (8:92 MeOH:CHCl3) afforded 2 (54 mg, 20%)
as a brown solid; Rf 0.40 (8:92 MeOH:CHCl3); mp 104-106
°C; IR (Nujol) 1661, 1632, 1594 cm-1; 1H NMR (CDCl3) δ 8.08
(m, 2H), 7.78 (m, 2H), 7.03 (t, 1H), 5.01 (m, 2H, J P,H ) 7.3
Hz), 3.68 (t, 4H), 3.23 (dt, 4H), 2.89 (bs, 2H); 31P NMR (CDCl3)
δ 15.00; FAB MS (C15H17N2O5P) calcd 337.0953, obsd 337.0957.
2-(1,4-Dim eth oxyn a p h th yl)m eth yl N,N-Bis(2-br om o-
eth yl)p h osp h or od ia m id a te (13a ). Lithium bis(trimethyl-
silyl)amide (6.05 mL, 6.05 mmol, 1 M in THF) was added
dropwise via syringe to a solution of 11a (1.20 g, 5.50 mmol)
in THF (20 mL) at -78 °C under argon. The resulting solution
was stirred for 5 min and then added dropwise to a solution
of 8 (2.29 g, 6.60 mmol) in THF (50 mL) at -78 °C. The
reaction mixture was stirred at -78 °C for 1.5 h and then
warmed to -20 °C. Gaseous ammonia was passed through the
reaction mixture for 10 min. The mixture was stirred for 10
min, aqueous HCl (2%, 70 mL) was added and the mixture
was extracted with EtOAc (3×). The combined organic layers
were washed with saturated NaCl (2×), dried (MgSO4), filtered
and evaporated. Column chromatography of the crude product
(70:30 EtOAc:hexanes) afforded 13a (1.20 g, 43%) as a white
solid: Rf 0.33 (70:30 EtOAc:hexanes); mp 112-113 °C; 1H
NMR (CDCl3) δ 8.23 (dd, 1H), 8.06 (dd, 1H), 7.54 (m, 2H), 6.84
(s, 1H), 5.26 (m, 2H, J P,H ) 8.1 Hz), 4.01 (s, 3H), 3.94 (s, 3H),
3.50 (m, 8H), 2.78 (bs, 2H); 31P NMR (CDCl3) δ 15.67.
2-(3-Th iom eth yl-1,4-d im eth oxyn a p h th yl)m eth yl N,N-
bis(2-ch lor oet h yl)p h osp h or od ia m id a t e (13e) was pre-
pared from 11c as described for the synthesis of 13c (0.27-
mmol scale). Column chromatography of the crude product
(EtOAc) afforded 13e (77 mg, 61%) as a yellow oil: Rf 0.22
1
(EtOAc); H NMR (CDCl3) d 8.12 (m, 2H), 7.57 (m, 2H), 5.48
(d, 2H, J P,H ) 4.9 Hz), 4.04 (s, 3H), 4.00 (s, 3H), 3.64 (t, 4H),
3.49 (dt, 4H), 2.73 (bs, 2H), 2.51 (s, 3H); 31P NMR (CDCl3) δ
14.62.
2-(3-Br om o-1,4-d im eth oxyn a p h th yl)m eth yl N,N-bis(2-
ch lor oeth yl)p h osp h or od ia m id a te (13f) was prepared from
11d as described for the synthesis of 13c (0.91-mmol scale).
Column chromatography of the crude product (EtOAc) afforded
13f (353 mg, 78%) as a white solid: Rf 0.17 (EtOAc); mp 112-
2-(3-Meth yl-1,4-d im eth oxyn a p h th yl)m eth yl N,N-bis(2-
br om oeth yl)p h osp h or od ia m id a te (13b) was prepared from
11b as described for the synthesis of 13a (2.07-mmol scale).
Column chromatography of the crude product (2:98 MeOH:
EtOAc) afforded 13b (480 mg, 44%) as a viscous yellow oil: Rf
0.63 (2:98 MeOH:EtOAc); 1H NMR (CDCl3) δ 8.09 (d, 2H), 7.41
(m, 2H), 5.31 (m, 2H, J P,H ) 7.1 Hz), 3.98 (s, 3H), 3.89 (s, 3H),
3.48 (m, 8H), 2.77 (bs, 2H), 2.53 (s, 3H); 31P NMR (CDCl3) δ
15.32.
1
114 °C; H NMR (CDCl3) δ 8.12 (m, 2H), 7.60 (m, 2H), 5.40
(m, 2H, J P,H ) 5.9 Hz), 4.02 (s, 3H), 4.00 (s, 3H), 3.66 (t, 4H),
3.48 (dt, 4H), 2.82 (bs, 2H); 31P NMR (CDCl3) δ 14.38.
2-(1,4-Na p h th oqu in on yl)m eth yl N,N-bis(2-ch lor oeth -
yl)p h osp h or od ia m id a te (3c) was prepared from 13c as
described for the synthesis of 3a (1.14-mmol scale). Column
chromatography of the crude product (6:94 MeOH:CHCl3)
afforded 3c (360 mg, 81%) as a yellow solid: Rf 0.45 (6:94
MeOH:CHCl3); mp 105-107 °C; IR (Nujol) 1664, 1630, 1589
2-(1,4-Na p h th oqu in on yl)m eth yl N,N-Bis(2-br om oeth -
yl)p h osp h or od ia m id a te (3a ). Ceric ammonium nitrate (1.83
g, 3.33 mmol) in H2O (15 mL) was added in portions over 15
min to a solution of 13a (680 mg, 1.33 mmol) in CH3CN (60
mL). The reaction was stirred at room temperature for 1 h
and extracted with CHCl3 (3×). The combined organic layers
were dried (MgSO4), filtered and evaporated. Column chro-
matography of the crude product (2:98 MeOH:EtOAc) afforded
3a (550 mg, 86%) as a yellow solid: Rf 0.51 (2:98 MeOH:
1
cm-1; H NMR (CDCl3) δ 8.09 (m, 2H), 7.77 (m, 2H), 7.03 (t,
1H), 5.05 (m, 2H, J P,H ) 7.1 Hz), 3.68 (t, 4H), 3.54 (m, 4H),
3.01 (bs, 2H); 31P NMR (CDCl3) δ 16.42. Anal. (C15H17Cl2N2O4P)
C, H, N.
2-(3-Met h yl-1,4-n a p h t h oq u in on yl)m et h yl N,N-b is(2-
ch lor oeth yl)p h osp h or od ia m id a te (3d ) was prepared from
13d as described for the synthesis of 3a (1.01-mmol scale).
Column chromatography of the crude product (3:97 MeOH:
CHCl3) afforded 3d (289 mg, 71%) as a yellow solid: Rf 0.16
(3:97 MeOH:CHCl3); mp 129-130 °C; IR (Nujol) 1665, 1660,
EtOAc); mp 118-119 °C; IR (Nujol) 1664, 1627, 1591 cm-1
;
1H NMR (CDCl3) δ 8.10 (m, 2H), 7.77 (m, 2H), 7.03 (t, 1H),
5.05 (m, 2H, J P,H ) 7.0 Hz), 3.55 (m, 8H), 2.98 (bs, 2H); 31P
NMR (CDCl3) δ 16.18. Anal. (C15H17Br2N2O4P) C, H, N.
1628, 1594 cm-1 1H NMR (CDCl3) δ 8.11 (m, 2H), 7.76 (m,
;
2H), 5.06 (m, 2H, J P,H ) 7.3 Hz), 3.66 (t, 4H), 3.47 (dt, 4H),
3.02 (bs, 2H), 2.34 (s, 3H); 31P NMR (CDCl3) δ 14.27. Anal.
(C16H19Cl2N2O4P) C, N, H.
2-(3-Met h yl-1,4-n a p h t h oq u in on yl)m et h yl N,N-b is(2-
br om oeth yl)p h osp h or od ia m id a te (3b) was prepared from
13b as described for the synthesis of 3a (0.90-mmol scale).
Column chromatography of the crude product (6:94 MeOH:
CHCl3) afforded 3b (230 mg, 52%) as a yellow solid: Rf 0.28
(6:94 MeOH:CHCl3); mp 117-119 °C; IR (Nujol) 1662, 1626,
2-(3-Th iom eth yl-1,4-n a p h th oqu in on yl)m eth yl N,N-bis-
(2-ch lor oeth yl)p h osp h or od ia m id a te (3e) was prepared
from 13e as described for the synthesis of 3a (0.287-mmol
scale). Column chromatography of the crude product (2:98
MeOH:CHCl3) afforded 3e (72 mg, 57%) as a viscous orange
oil: Rf 0.29 (2:98 MeOH:CHCl3); IR (Nujol) 1666, 1643, 1588,
1542 cm-1; 1H NMR (CDCl3) δ 8.10 (m, 2H), 7.75 (m, 2H), 5.21
(m, 2H, J P,H ) 6.1 Hz), 3.66 (t, 4H), 3.46 (dt, 4H), 2.97 (bs,
1594, 1568 cm-1 1H NMR (CDCl3) δ 8.12 (m, 2H), 7.76 (m,
;
2H), 5.06 (m, 2H, J P,H ) 7.6 Hz), 3.50 (m, 8H), 3.02 (bs, 2H),
2.34 (s, 3H); 31P NMR (CDCl3) δ 15.72; FAB MS (C16H19
Br2N2O4P) calcd 492.9527, obsd 492.9542.
-
2H), 2.74 (s, 3H); 31P NMR (CDCl3) δ 15.25. Anal. (C16H19
Cl2N2O4PS) C, H, N.
-
2-(1,4-Dim eth oxyn a p h th yl)m eth yl N,N-Bis(2-ch lor o-
eth yl)p h osp h or od ia m id a te (13c). Lithium bis(trimethylsi-
lyl)amide (2.50 mL, 2.50 mmol, 1.0 M in THF) was added
dropwise via syringe to a solution of 11a (500 mg, 2.29 mmol)
2-(3-Br om o-1,4-n a p h t h oq u in on yl)m et h yl N,N-Bis(2-
ch lor oeth yl)p h osp h or od ia m id a te (3f). Silver(II) oxide (300

3164 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 16
Flader et al.
mg, 2.4 mmol) was added in one portion to a solution of 13f
(200 mg, 0.40 mmol) in THF (15 mL) at room temperature.
Nitric acid (6 N, 2 mL) was added to this suspension. The
mixture was stirred for 20 min at room temperature, H2O (5
mL) was added and the mixture was extracted with CHCl3
(3×). The combined organic layers were washed with saturated
NaCl and NaHCO3 (1 M), dried (MgSO4), filtered and evapo-
rated. Column chromatography of the crude product (2:98
MeOH:CHCl3 to remove impurities, then 6:94 MeOH:CHCl3)
afforded 3f (156 mg, 83%) as a yellow solid: Rf 0.36 (6:94
MeOH:CHCl3); mp 134-135 °C; IR (Nujol) 1680, 1675, 1659,
2,3-Din itr o-1,4-d im eth oxyben zen e (16) was prepared as
described.15 1,4-Dimethoxybenzene (3.00 g, 2.17 mmol) was
added slowly with vigorous stirring to concentrated HNO3 (27
mL) at 0 °C. The thick, yellow suspension was warmed to room
temperature and stirred for 1 h. The mixture was then heated
to 90 °C and stirred at 90 °C for 1 h, cooled to room
temperature and poured into ice water (125 mL). The product
was collected by filtration, washed with water, and dried to
give 4.39 g (89%) of a bright yellow solid. The product was
determined to be an 8:1 mixture of 16 and 2,5-dinitro-1,4-
1
dimethoxybenzene by H NMR. The crude product was used
1605, 1589 cm-1
;
1H NMR (CDCl3) δ 8.18 (m, 2H), 7.80 (m,
without further purification in the subsequent reaction: Rf
1
2H), 5.21 (m, 2H, J P,H ) 6.5 Hz), 3.66 (t, 4H), 3.48 (dt, 4H),
0.20 (50:50 EtOAc:hexanes); H NMR (CDCl3) δ 7.20 (s, 2H),
2.97 (bs, 2H); 31P NMR (CDCl3) δ 15.32. Anal. (C15H16
BrCl2N2O4P) C, H, N.
-
3.94 (s, 6H).
4,7-Dim eth oxy-2-h yd r oxym eth ylben zim id a zole (17).
Stannous chloride (22.4 g, 0.118 mol) was added in portions
to a suspension of 16 (3.00 g, 0.013 mol) in concentrated HCl
(33 mL) at 0 °C. The ice bath was removed and the reaction
mixture became warm. The temperature was maintained
below 100 °C by occasional application of an ice bath. When
the temperature of the mixture fell to 70 °C, the mixture was
heated to 90-100 °C for 2 h. The pale yellow mixture was
cooled to room temperature, and water (100 mL) and glycolic
acid (2.60 g, 0.034 mol) were added. The mixture was refluxed
overnight. The clear, pale green solution was cooled to room
temperature, slowly neutralized with saturated Na2CO3, and
extracted with EtOAc (6×). The combined organic layers were
washed with saturated NaCl (2×), dried (MgSO4), filtered and
evaporated. Column chromatography of the crude product (10:
90 MeOH:EtOAc) afforded 17 (2.11 g, 78%) as an amorphous
gray solid: Rf 0.41 (10:90 MeOH:EtOAc); 1H NMR (DMSO-
d6) δ 6.56 (s, 2H), 4.57 (s, 2H), 3.85 (s, 6H).
4,7-Dim et h oxy-2-h yd r oxym et h yl-1-m et h ylb en zim id -
a zole (18). Lithium bis(trimethylsilyl)amide (10.1 mL, 10.1
mmol, 1 M in THF) was added dropwise via syringe to a
solution of 17 (1.00 g, 4.80 mmol) in THF (50 mL) at 0 °C under
argon. The mixture was warmed to room temperature and
stirred for 20 min. A solution of methyl iodide (0.33 mL, 5.3
mmol) in THF (20 mL) was added dropwise via syringe. The
solution was stirred for 7 h, H2O (40 mL) was added and the
mixture was extracted with EtOAc (4×). The combined organic
layers were washed with saturated NaCl (3×), dried (MgSO4),
filtered and evaporated. Column chromatography of the crude
product (10:90 MeOH:EtOAc) afforded 18 (0.98 g, 92%) as a
brown solid: Rf 0.50 (10:90 MeOH:EtOAc); mp 153-155 °C;
1H NMR (CDCl3) δ 6.57 (d, 1H), 6.50 (d, 1H), 4.86 (s, 2H), 4.02
(s, 3H), 3.96 (s, 3H), 3.89 (s, 3H).
2-(4,7-Dim eth oxy-1-m eth ylben zim idazolyl)m eth yl N,N-
Bis(2-br om oeth yl)ph osph or odiam idate (19a). Lithium bis-
(trimethylsilyl)amide (2.80 mL, 2.80 mmol, 1 M in THF) was
added dropwise via syringe to a solution of 18 (560 mg, 2.52
mmol) in CH2Cl2 (10 mL) and THF (4 mL) at -78 °C under
argon. The resulting solution was stirred for 5 min and then
added via syringe to a solution of 8 (1.05 g, 3.02 mmol) in THF
(28 mL). The mixture was stirred at -78 °C for 1.5 h and then
warmed to -20 °C. Gaseous ammonia was passed through the
solution for 7 min and the cloudy mixture was stirred for 8
min. Hydrochloric acid (2%, 35 mL) was added and the mixture
was extracted with EtOAc (4×). The combined organic layers
were washed with saturated Na2CO3 (2×) and saturated NaCl
(2×), dried (MgSO4), filtered and evaporated. Column chro-
matography of the crude product (68:29:3 CHCl3:acetone:
MeOH) afforded 19a (610 mg, 47%) as an amorphous tan
solid: Rf 0.24 (68:29:3 CHCl3:acetone:MeOH); 1H NMR (CDCl3)
δ 6.60 (d, 1H), 6.52 (d, 1H), 5.26 (m, 2H, J P,H ) 8.2 Hz), 4.10
(s, 3H), 3.96 (s, 3H), 3.90 (s, 3H), 3.47 (m, 8H), 3.13 (bs, 2H);
31P NMR (CDCl3) δ 16.27.
2-Acetoxym eth yl-1,4-d im eth oxyn a p h th a len e (14). Tri-
ethylamine (0.35 mL, 2.52 mmol) was added with stirring to
a solution of 11a (500 mg, 2.29 mmol) in CH2Cl2 (25 mL) in a
room-temperature water bath under argon. Acetyl chloride
(0.18 mL, 2.52 mmol) was added dropwise, and the solution
was stirred at room temperature for 3 h and concentrated
under reduced pressure. The residue was resuspended in
EtOAc and the salts were separated. Column chromatography
of the crude product (1:9 EtOAc:hexanes) afforded 14 (540 mg,
91%) as a colorless oil: Rf 0.40 (1:9 EtOAc:hexanes); 1H NMR
(CDCl3) δ 8.23 (dd, 1H), 8.07 (dd, 1H), 7.54 (m, 2H), 6.76 (s,
1H), 5.34 (s, 2H), 3.99 (s, 3H), 3.94 (s, 3H), 2.13 (s, 3H).
2-Acetoxym eth yl-1,4-n a p h th oqu in on e (1). Ceric am-
monium nitrate (2.84 g, 5.19 mmol) in H2O (25 mL) was added
in portions over 15 min to a solution of 14 (540 mg, 2.07 mmol)
in CH3CN (30 mL) at room temperature. The mixture was
stirred at room temperature for 30 min and extracted with
CHCl3 (4×). The combined organic layers were dried (MgSO4),
filtered and evaporated. Column chromatography of the crude
product (3:7 EtOAc:hexanes) afforded 1 (420 mg, 88%) as a
yellow solid: Rf 0.66 (3:7 EtOAc:hexanes); mp 107-109 °C
(lit.15 mp 109-110 °C); IR (Nujol) 1739, 1659, 1630, 1593 cm-1
;
1H NMR (CDCl3) δ 8.10 (m, 2H), 7.77 (m, 2H), 6.91 (t, 1H, J )
2 Hz), 5.15 (d, 2H, J ) 2 Hz), 2.20 (s, 3H).
2-(1,4-Dim eth oxyn a p h th yl)m eth yl Bis[N-(2-ch lor oeth -
yl)]ph osph or odiam idate (15). Phosphorus oxychloride (0.043
mL, 0.46 mmol) was added to a suspension of N-(2-chloro-
ethyl)amine hydrochloride (112 mg, 0.96 mmol) in CH2Cl2 (10
mL) at -78 °C under argon. Diisopropylethylamine (0.34 mL,
1.92 mmol) was added dropwise and the reaction mixture was
warmed to room temperature over 2 h and stirred for 7 h.
n-Butyllithium (0.18 mL, 0.46 mmol, 2.5 M in hexanes) was
added to a solution of a 11a (100 mg, 0.46 mmol) in CH2Cl2 (5
mL) in a second flask at -78 °C under argon. The resulting
solution was stirred for 15 min. The phosphoryl chloride
solution was added via syringe to the alkoxide solution with
vigorous stirring at -78 °C. The reaction mixture was slowly
warmed to room temperature, stirred overnight, and then
concentrated under reduced pressure until the volume was
reduced to one-third. The salts were separated and the
crude product was purified by column chromatography (78:22
CHCl3:acetone to remove impurities, then 67:33 CHCl3:
acetone) to afford 15 (62.0 mg, 32%, 87% based on recovered
1
11a ) as a pale yellow oil: Rf 0.31 (78:22 CHCl3:acetone); H
NMR (CDCl3) δ 8.24 (dd, 1H), 8.06 (dd, 1H), 7.55 (m, 2H), 6.84
(s, 1H), 5.25 (d, 2H, J P,H ) 7.9 Hz), 4.01 (s, 3H), 3.94 (s, 3H),
3.59 (t, 4H), 3.28 (dt, 4H), 3.04 (bs, 2H); 31P NMR (CDCl3) δ
14.72.
2-(1,4-Na p h th oqu in on yl)m eth yl bis[N-(2-ch lor oeth yl)]-
p h osp h or od ia m id a te (5) was prepared from 15 as described
for the synthesis of 3a (0.37-mmol scale). Column chromatog-
raphy of the crude product (78:22 CHCl3:acetone to remove
impurities, then 67:33 CHCl3:acetone) afforded 5 (42.4 mg,
74%) as a yellow solid: Rf 0.24 (67:33 CHCl3:acetone); mp 109-
2-(4,7-Dim eth oxy-1-m eth ylben zim idazolyl)m eth yl N,N-
bis(2-ch lor oeth yl)p h osp h or od ia m id a te (19b) was pre-
pared from 18 and bis(2-chloroethyl)phosphoramidic dichloride
as described for the synthesis of 19a (1.80 mmol). Column
chromatography of the crude product (68:29:3 CHCl3:acetone:
MeOH) afforded 19b (390 mg, 51%, 62% based on recovered
18, 70 mg) as a tan foam: Rf 0.30 (68:29:3 CHCl3:acetone:
1
110 °C; IR (Nujol) 1662, 1633, 1594 cm-1; H NMR (CDCl3) δ
8.10 (m, 2H), 7.77 (m, 2H), 7.03 (t, 1H, J ) 1.9 Hz), 5.04 (dd,
2H, J ) 1.9 and 6.8 Hz), 3.65 (t, 4H), 3.36 (dt, 4H), 3.20 (bs,
2H); 31P NMR (CDCl3) δ 14.83. Anal. (C15H17Cl2N2O4P) C, H,
N.

Quinone Phosphorodiamidate Prodrugs
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 16 3165
MeOH); 1H NMR (CDCl3) δ 6.59 (d, 1H), 6.52 (d, 1H), 5.26 (m,
2H, J P,H ) 8.2 Hz), 4.10 (s, 3H), 3.96 (s, 3H), 3.90 (s, 3H), 3.64
(t, 4H), 3.46 (dt, 4H), 3.05 (bs, 2H); 31P NMR (CDCl3) δ 16.43.
1H NMR (CDCl3) δ 8.10 (m, 2H), 7.77 (m, 2H), 7.03 (t, 1H, J )
2.0 Hz), 5.04 (dd, 2H, J ) 2.0 and 6.4 Hz), 3.49 (m, 8H), 2.78
(d, 6H, J ) 9.7 Hz); 31P NMR (CDCl3) δ 18.00. Anal. (C17H21
-
Br2N2O4P) C, H, N.
2-(1-Meth yl-4,7-ben zim id a zolequ in on yl)m eth yl N,N-
bis(2-br om oeth yl)p h osp h or od ia m id a te (4a ) was prepared
as described for 3f using 19a (0.194-mmol scale). Column
chromatography of the crude product (68:29:3 CHCl3:acetone:
MeOH to remove impurities, then 53:42:5 CHCl3:acetone:
MeOH) afforded 4a (62.7 mg, 67%) as a yellow foam: Rf 0.18
2-(3-Meth yl-1,4-n a p h th oqu in on yl)m eth yl bis[N-m eth -
yl-N-(2-br om oeth yl)]p h osp h or od ia m id a te (6b) was pre-
pared from 20b as described for the synthesis of 3a (0.235-
mmol scale). Column chromatography of the crude product (3:
97 MeOH:CH2Cl2) afforded 6b (99 mg, 81%) as a yellow oil:
(68:29:3 CHCl3:acetone:MeOH); IR (Nujol) 1665, 1521 cm-1
;
Rf 0.20 (3:97 MeOH:CH2Cl2); IR (neat) 1663, 1626, 1595 cm-1
;
1H NMR (CDCl3) δ 6.72 (d, 1H), 6.65 (d, 1H), 5.24 (m, 2H, J P,H
) 8.5 Hz), 4.07 (s, 3H), 3.50 (m, 8H), 3.16 (bs, 2H); 31P NMR
(CDCl3) δ 16.53; FAB MS (C13H17Br2N4O4P) calcd 482.9433,
obsd 482.9432.
1H NMR (CDCl3) δ 8.12 (m, 2H), 7.75 (m, 2H), 5.04 (d, 2H, J
) 6.4 Hz), 3.43 (m, 8H), 2.71 (d, 6H, J ) 9.7 Hz), 2.35 (s, 3H);
31P NMR (CDCl3) δ 17.77. Anal. (C18H23Br2N2O4P) C, H, N.
2-(1,4-Na p h t h oq u in on yl)m et h yl b is[N-m et h yl-N-(2-
ch lor oeth yl)]p h osp h or od ia m id a te (6c) was prepared from
20c as described for the synthesis of 3a (0.29-mmol scale).
Column chromatography of the crude product (2:98 MeOH:
EtOAc) afforded 6c (105 mg, 87%) as a brown oil: Rf 0.55 (2:
2-(1-Meth yl-4,7-ben zim id a zolequ in on yl)m eth yl N,N-
bis(2-ch lor oeth yl)p h osp h or od ia m id a te (4b) was prepared
from 19b as described for the synthesis of 3f (0.47-mmol scale).
Column chromatography of the crude product (68:29:3 CHCl3:
acetone:MeOH) afforded 4b (146 mg, 79%) as a yellow foam:
Rf 0.18 (68:29:3 CHCl3:acetone:MeOH); IR (neat) 1665, 1591,
1518 cm-1; 1H NMR (CDCl3) δ 6.72 (d, 1H), 6.65 (d, 1H), 5.22
(m, 2H, J P,H ) 8.3 Hz), 4.07 (s, 3H), 3.66 (t, 4H), 3.49 (dt, 4H),
1
98 MeOH:EtOAc); IR (neat) 1664, 1633, 1595 cm-1; H NMR
(CDCl3) δ 8.10 (m, 2H), 7.77 (m, 2H), 7.03 (t, 1H, J ) 2.0),
5.03 (dd, 2H, J ) 2.0 and 6.4 Hz), 3.66 (t, 4H), 3.42 (m, 4H),
2.78 (d, 6H, J ) 9.9 Hz); 31P NMR (CDCl3) δ 18.21 ppm. Anal.
(C17H21Cl2N2O4P) C, H, N.
3.09 (bs, 2H); 31P NMR (CDCl3) δ 16.74; FAB MS (C13H17
Cl2N4O4P) calcd 395.0445, obsd 395.0461.
-
En zym e Kin etics. The DT-diaphorase activity of the
synthesized quinone compounds was determined using a
standard coupled assay according to a modification of the
procedure described by Ernster.18 The reactions were moni-
tored using Kinetics software by Varian, version 1.0, and a
CARY 3 UV-visible spectrophotometer. Reaction mixtures,
totaling 1 mL in volume, contained Tris buffer (0.05 M, pH
7.6) and dithiothreitol (0.01 mM), NADH (1 mM), purified
human enzyme (4.8 × 10-4 mg), cytochrome c (55 µM) and a
suitable concentration of the quinone in DMSO (10 µL). The
reactions were run in triplicate at 25 °C and started by the
addition of the quinone. They were monitored by recording an
increase in absorbance at 550 nm due to the reduction of
cytochrome c, and appropriate controls were performed with-
out the addition of enzyme to determine the background rates
for the reactions. Rates of reduction were measured from the
initial linear portion of the reaction curve and the kinetic
parameters were determined using a Hanes plot. An extinction
coefficient of 1.85 × 104 M-1 cm-1 for cytochrome c was used
in the calculations.
Mea su r em en t of DT-Dia p h or a se, NADP H:Cytoch r om e
P -450 Red u cta se, a n d NADH:Cytoch r om e b5 Red u cta se
Activity in HT-29 a n d BE Cells. S9 supernatants were
prepared from HT-29 and BE cell lines using a procedure
outlined by Fitzsimmons et al.,20 except that the cell pellets
were resuspended immediately. Protein concentration was
determined using a standard Bradford assay.26 Enzyme activi-
ties were measured using a modification of the procedure
described by Fitzsimmons et al.20 The same reaction mixtures
were used as described above, except that menadione (2 µM)
was used as the substrate and the assays were run at 37 °C.
Using NADH as cofactor, the enzyme activity present when
dicumarol (10 µM) was added as an inhibitor of DT-diaphorase
was attributed to cytochrome b5 reductase, and the activity
that was inhibited by dicumarol was attributed to DT-
diaphorase. Using NADPH as cofactor, the enzyme activity
present when dicumarol was added as an inhibitor of DT-
diaphorase was attributed to cytochrome P-450 reductase.20
Enzyme activity is expressed as nmol cytochrome c reduced/
min/mg protein.
2-(1,4-Dim eth oxyn a p h th yl)m eth yl bis[N-m eth yl-N-(2-
br om oeth yl)]p h osp h or od ia m id a te (20a ). A solution of 11a
(100 mg, 0.46 mmol) in CH2Cl2 (2 mL) and CH3CN (2 mL) was
cooled to -70 °C and stirred under argon. Phosphorus trichlo-
ride (0.23 mL, 0.46 mmol, 2 M in CH2Cl2) was added dropwise
followed by the dropwise addition of diisopropylethylamine
(0.08 mL, 0.46 mmol). The reaction mixture was stirred for
25 min at -70 °C. A solution of N-methyl-N-(2-bromoethyl)-
amine hydrobromide10 (200 mg, 0.92 mmol) in CH3CN (2.5 mL)
was added slowly via syringe, followed by additional diisopro-
pylethylamine (0.32 mL, 1.84 mmol). The temperature was
raised to -60 to -50 °C and the reaction was stirred for 1.25
h. tert-Butyl hydroperoxide (1.1 mL, 5-6 M in decane) was
added, and the reaction mixture was warmed to -40 to -20
°C and stirred for 30 min. Water (3 mL) was added, and the
mixture was warmed to room temperature and extracted with
CH2Cl2 (4×). The combined organic layers were washed with
H2O and saturated NaCl, dried (MgSO4), filtered and evapo-
rated. Column chromatography of the crude product (2:98
MeOH:EtOAc) afforded 20a (190 mg, 77%) as a pale yellow
oil: Rf 0.33 (2:98 MeOH:EtOAc); 1H NMR (CDCl3) δ 8.24 (dd,
1H), 8.06 (dd, 1H), 7.54 (m, 2H), 6.87 (s, 1H), 5.24 (d, 2H, J )
7.4 Hz), 4.01 (s, 3H), 3.93 (s, 3H), 3.43 (m, 8H), 2.71 (d, 6H, J
) 9.7 Hz); 31P NMR (CDCl3) δ 17.60.
2-(3-Meth yl-1,4-dim eth oxyn aph th yl)m eth yl bis[N-m eth -
yl-N-(2-br om oeth yl)]p h osp h or od ia m id a te (20b) was pre-
pared from 11b and N-methyl-N-(2-bromoethyl)amine hydro-
bromide10 as described for the synthesis of 20a (0.43 mmol).
Column chromatography of the crude product (2:98 MeOH:
EtOAc) afforded 20b (135 mg, 57%) as a yellow oil: Rf 0.37
(2:98 MeOH:EtOAc); 1H NMR (CDCl3) δ 8.09 (dd, 2H), 7.52
(m, 2H), 5.28 (d, 2H, J ) 5.8 Hz), 3.97 (s, 3H), 3.89 (s, 3H),
3.41 (m, 8H), 2.67 (d, 6H, J ) 9.7 Hz), 2.53 (s, 3H); 31P NMR
(CDCl3) δ 17.28.
2-(1,4-Dim eth oxyn a p h th yl)m eth yl bis[N-m eth yl-N-(2-
ch lor oeth yl)]ph osph or odiam idate (20c) was prepared from
11a and N-methyl-N-(2-chloroethyl)amine hydrochloride10 as
described for the synthesis of 20a (0.458 mmol). Column
chromatography of the crude product (2:98 MeOH:EtOAc)
afforded 20c (132 mg, 64%) as a pale yellow oil: Rf 0.33 (2:98
MeOH:EtOAc); 1H NMR (CDCl3) δ 8.24 (dd, 1H), 8.06 (dd, 1H),
7.54 (m, 2H), 6.88 (s, 1H), 5.24 (d, 2H, J ) 7.3 Hz), 4.00 (s,
3H), 3.93 (s, 3H), 3.63 (t, 4H), 3.37 (dt, 4H), 2.72 (d, 6H, J )
9.6 Hz); 31P NMR (CDCl3) δ 17.91.
31P NMR Kin etics. The quinone prodrug was dissolved in
CH3CN (0.3 mL, THF for 3a ) and the activating agent
(glutathione, DDTC or sodium dithionite) was dissolved in
cacodylate buffer (0.4 mL, 0.4 M, pH 7.7). The buffer solution
was added to the organic solution and the pH of the mixture
was adjusted to ∼7.4. The reaction mixture was transferred
to a 5-mm NMR tube, and the data acquisition was started
(pulse delay 30 µs). Spectra were taken every 2.5 min for 0.5
h, then every 5 min for 0.5 h, then every 10 min for 1 h, and
time points for each spectrum were assigned from the initiation
of the reaction. Chemical shifts are reported relative to the
2-(1,4-Na p h t h oq u in on yl)m et h yl b is[N-m et h yl-N-(2-
br om oeth yl)]p h osp h or od ia m id a te (6a ) was prepared from
20a as described for the synthesis of 3a (0.372-mmol scale).
Column chromatography of the crude product (2:98 MeOH:
CH2Cl2) afforded 6a (164 mg, 87%) as a viscous brown oil: Rf
0.16 (2:98 MeOH:CH2Cl2); IR (neat) 1664, 1632, 1595 cm-1
;

3166 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 16
Flader et al.
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Cytotoxicity Stu d ies in Vitr o: HT-29 a n d BE Cells. A
modification of the procedure described by Miribelli et al.27 was
used to determine the clonogenic survival of the cells. HT-29
and BE cells in exponential growth were suspended in un-
supplemented Eagles MEM medium (10 mL) at a final density
of 1.7-2 × 105 cells/mL. Unsupplemented medium contains
MEM (Gibco) and HEPES (0.02 M). The drug stock solutions
(0.1-40 mM) were prepared using either ethanol or dimethyl
sulfoxide as solvent. The maximum amount of DMSO or
ethanol used in the drug treatments was 1% of the final
volume. Appropriate volumes (6.5-100 µL) of the drug stock
solution were added to five vials of the cell suspensions, to
give five different final drug concentrations, and 100 µL of
solvent was added to a sixth vial for a control. The treated
cells were incubated for 2 h (37 °C, 5% CO2). The cells were
spun down and rinsed three times with supplemented medium
(3 mL) and then diluted in 5 mL of supplemented medium.
Supplemented medium was prepared by adding fetal bovine
serum (10%), gentamicin (0.05 mg/mL), l-glutamine (0.03 mg/
mL), and sodium pyruvate (0.1 mM) to unsupplemented
medium. The cells were counted using a Coulter Counter,
plated at 2-3 different densities for each drug concentration,
and incubated for 10 days. The colonies were stained with 0.5%
crystal violet in 95% ethanol, and those colonies comprised of
50 or more cells were counted using a dissecting microscope
and pen-style counter. The LC99 of each compound was
determined by plotting the log surviving fraction vs drug
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Ack n ow led gm en t. We thank Dr. Shiuan Chen
(City of Hope Medical Center, CA) for providing purified
human DT-diaphorase and Dr. David Ross (University
of Colorado) for providing the BE cell line. The as-
sistance of the Purdue Cancer Center Drug Evaluation
Facility in carrying out the xenograft experiments is
appreciated. Financial support from Grants R01 CA34619
and T32 GM08298 is also gratefully acknowledged.
(12) J acob, P., III; Callery, P. S.; Shulgin, A. T.; Castagnoli, N., J r.
A convenient synthesis of quinones from hydroquinone dimethyl
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