Nitrobenzyl-based photosensitive phosphoramide mustards: Synthesis and photochemical properties of potential prodrugs for cancer therapy

Article abstract of DOI:10.1021/jo961861m

Several nitrobenzyl-based photosensitive phosphoramide mustards were synthesized. The nitrobenzyl moiety was structurally varied to find the most promising prodrug candidates in respect to photorelease and activity of the alkylating species. The synthesis of these compounds proved to be applicable even in regard to compounds with additional functionalization. The target molecules 13a,b to 14 exhibited the expected red shift in their absorption spectra maximum compared to the parent nitrobenzyl moiety. As seen by UV and ³¹P NMR spectroscopy, the phosphoramide mustard was quickly liberated upon irradiation with mercury arc lamps. Assaying the structurally different prodrugs on their alkylating activity showed that compounds 13b and 14, derived from secondary benzyl alcohols, are promising prodrug candidates. Their water solubility and the possibility of attaching macromolecules are encouraging vis-a-vis future investigations on their in vitro cytotoxicity.

Full text of DOI:10.1021/jo961861m

2434
J . Org. Chem. 1998, 63, 2434-2441
Articles
Nitr oben zyl-Ba sed P h otosen sitive P h osp h or a m id e Mu sta r d s:
Syn th esis a n d P h otoch em ica l P r op er ties of P oten tia l P r od r u gs for
Ca n cer Th er a p y
Robert Reinhard and Brigitte F. Schmidt*
Center for Light Microscope Imaging and Biotechnology, Carnegie Mellon University, 4400 Fifth Avenue,
Pittsburgh, Pennsylvania 15213
Received October 1, 1996 (Revised Manuscript Received February 23, 1998)
Several nitrobenzyl-based photosensitive phosphoramide mustards were synthesized. The ni-
trobenzyl moiety was structurally varied to find the most promising prodrug candidates in respect
to photorelease and activity of the alkylating species. The synthesis of these compounds proved to
be applicable even in regard to compounds with additional functionalization. The target molecules
13a ,b to 14 exhibited the expected red shift in their absorption spectra maximum compared to the
parent nitrobenzyl moiety. As seen by UV and 31P NMR spectroscopy, the phosphoramide mustard
was quickly liberated upon irradiation with mercury arc lamps. Assaying the structurally different
prodrugs on their alkylating activity showed that compounds 13b and 14, derived from secondary
benzyl alcohols, are promising prodrug candidates. Their water solubility and the possibility of
attaching macromolecules are encouraging vis-a`-vis future investigations on their in vitro
cytotoxicity.
In tr od u ction
been well studied, especially by Trentham and co-
workers.9 Initial applications involved “caged ATP”10 and
continued with the caging of a series of biologically
important molecules. Applications include modern tech-
niques such as fluorescent imaging,11 light-directed solid-
phase synthesis,12 and combinatorial chemistry.13
Photolabile (“caged”) compounds provide an important
tool in the investigation of many biological processes.1
Most functionalities occurring in biological molecules,
such as amines,2 alcohols,3 carboxylic acids,4 phosphates,5
and thiols,6 have been caged, and the feasibility of this
concept has been established by the reactivation of their
biological function after irradiation with light of ap-
propriate wavelength. A number of light-sensitive pro-
tective groups have been developed.1a,7 The nitrobenzyl
group has been shown to be among the most useful
members in this group of compounds, primarily due to
its ability to derivatize a number of functional groups,
the reliability of its photochemical reaction in different
systems, and the often straightforward synthesis of the
caged moiety.8 The mechanism of the intramolecular
redox process responsible for the decaging reaction has
Antiproliferative agents play a very important role in
cancer chemotherapy.14 Compounds with alkylating
activity remain among the most valuable in this field, in
particular the phosphoramide mustard-based agents
(e.g., cyclophosphamide).15 Most of the phosphoramide
mustard-derived reagents in and of themselves are
inactive “prodrugs”. They are activated by different
mechanisms that involve hydrolytic,16 bioreductive,17 and
(9) Walker, J . W.; Reid, G. P.; McCray, J . A.; Trentham, D. R. J .
Am. Chem. Soc. 1988, 110, 7170-7177.
(10) Kaplan, J . H.; Forbush, B., III; Hoffman, J . F. Biochemistry
1978, 17, 1929-1935.
* To whom correspondence should be addressed. E-mail: Bschmidt@
andrew.cmu.edu.
(11) Corrie, J . E. T.; Trentham, D. R. J . Chem. Soc., Perkin Trans.
1 1995, 1993-2000.
(1) For a recent and comprehensive overview see: (a) Bioorganic
Photochemistry, Vol. 2, Biological Applications of Photochemical
Switches; Morrison, H., Ed.; Wiley: New York, 1993. (b) Adams, S.
R.; Tsien, R. Y. Annu. Rev. Physiol. 1993, 55, 755-784. (c) Gurney, A.
M.; Lester, H. A. Physiol. Rev. 1987, 67, 583-617.
(12) (a) Pirrung, M. C.; Bradley, J .-C. J . Org. Chem. 1995, 60, 6270-
6276. (b) Greenberg, M. M.; Gilmore, J . L. J . Org. Chem. 1994, 59,
746-753.
(13) (a) Holmes, C. P.; J ones, D. G. J . Org. Chem. 1995, 60, 2318-
2319. (b) Borchardt, A.; Still, W. C. J . Am. Chem. Soc. 1994, 116, 373-
374.
(2) Mariott, G. Biochemistry 1994, 33, 9092-9097.
(3) Peng, L.; Goeldner, M. J . Org. Chem. 1996, 61, 185-191.
(4) (a) Gee, K. R.; Wieboldt, R.; Hess, G. P. J . Am. Chem. Soc. 1994,
116, 8366-8367. (b) Gee, K. R.; Niu, L.; Schaper, K.; Hess, G. P. J .
Org. Chem. 1995, 60, 4260-4263.
(14) For a recent overview see: Foye, W. O., Ed.; Cancer Chemo-
therapeutic Agents; ACS Professional Reference Books; American
Chemical Society: Washington, DC, 1995.
(15) Review: Friedman, O. M.; Myles, A.; Colvin, M. In Advances
in Cancer Chemotherapy; Rosowsky, A., Ed.; Marcel Dekker: New
York, 1979; pp 143-204.
(16) (a) Tietze, L. F.; Beller, M.; Fischer, R.; Loegers, M.; J aehde,
E.; Gluesenkamp, K.-H.; Rajewsky, M. F. Angew. Chem., Int. Ed. Engl.
1990, 29, 782-783. (b) Tietze, L. F.; Fischer, R.; Beller, M.; Seele, R.
Liebigs Ann. Chem. 1990, 151-157.
(17) (a) Mulcahy, R. T.; Gipp, J . J .; Schmidt, J . P.; J oswig, C.; Borch,
R. F. J . Med. Chem. 1994, 37, 1610-1615. (b) Firestone, A.; Mulcahy,
R. T.; Borch, R. F. J . Med. Chem. 1991, 34, 2933-2935.
(5) (a) Givens, R. S.; Kueper, W. L, III. Chem. Rev. 1993, 93, 55-
66. (b) Pirrung, M. C.; Shuey, S. W. J . Org. Chem. 1994, 59, 3890-
3897. (c) Walker, J . W.; Feeney, J .; Trentham, D. R. Biochemistry 1989,
28, 3272-3280.
(6) Mariott, G.; Heidecker, M. Biochemistry 1996, 35, 3170-3174.
(7) Review: Pillai, V. N. R. in Organic Photochemistry; Padwa, A.,
Ed.; Marcel Dekker: New York, 1987; Vol. 9, Chapter 3, pp 225-323.
(8) McCray, J . A.; Trentham, D. R. Annu. Rev. Biophys. Biophys.
Chem. 1989, 18, 239-270.
S0022-3263(96)01861-0 CCC: $15.00 © 1998 American Chemical Society
Published on Web 03/28/1998

Photosensitive Phosphoramide Mustards
J . Org. Chem., Vol. 63, No. 8, 1998 2435
Sch em e 1
Sch em e 3a
a
Reagents and conditions: (a) 2 HNTMS2, THF; (b) LHMDS,
THF, 0 °C; (c) THF, 0 °C then H+.
Sch em e 2
for cell-specific uptake, dyes for signaling, or macromol-
ecules for solid-phase chemistry in future applications.
Resu lts a n d Discu ssion
Syn th esis. We describe the synthesis of nitrobenzyl-
based phosphoramide mustard prodrugs that can be
activated by light. The feasibility of this approach, to
temporally inactivate phosphoramide mustard and re-
store its activity via irradiation, was tested with the
model compound 2-nitrobenzyl N,N-bis(2-chloroethyl)-
phosphordiamidate (10a ). The synthesis of this com-
pound is outlined in Scheme 3. N-Bis(2-chloroethyl)-
amidophosphoric acid dichloride (5), prepared from
phosphorus oxychloride and the appropriate amine hy-
drochloride,19 was converted to the bisamide 6 by the
reaction with 2 equiv of hexamethyldisilazane. The
2-nitrobenzyl alcohol 8a was deprotonated with lithium
bis(trimethylsilyl)amide (LHMDS) and reacted with in
situ generated 9 to yield, after final hydrolysis of the silyl
groups, the nitrobenzyl-protected phosphoramide mus-
tard 10a . After proving the successful photochemical
release of the phosphoramide mustard from our model
compound 10a (see next section), we developed a general
route for the synthesis of caged phosphoramide mustards
from higher functionalized 2-nitrobenzyl alcohols. We
reduced the commercially available 6-nitroveratralde-
hyde (7a ) with sodium borohydride to give the primary
benzyl alcohol 8b (Scheme 4). The secondary benzyl
alcohol 8c was prepared by stannous fluoride-mediated
alkylation of 2-nitrobenzaldehyde (7b) with allyl iodide.
Analogously, we synthesized the 5-hydroxy-2-nitrobenzyl
alcohols 11a and 11b from the commercially available
5-hydroxy-2-nitrobenzaldehyde (7c). Compounds 11a
and 11b opened a route to water-soluble caged phos-
phoramide mustards or prodrugs that could be linked to
other molecules such as dyes and proteins. The primary
alcohol 11a and the secondary alcohol 11b were alkylated
at the phenolic position by reaction with methyl bro-
biooxidative18 pathways. Interestingly, photochemical
activation of alkylating species has not been developed,
despite the potential advantage of controllable release.
This form of activation requires light of a particular
wavelength, typically λ > 340 nm.
The metabolism of cyclophosphamide (1)15 (Scheme 1)
suggests a strategy of introducing the caging moiety. To
get a light-sensitive prodrug candidate, we synthesized
nitrobenzyl esters of the phosphoric acid derivative 4a .
Irradiation of these compounds (type 10) should directly
lead to the buildup of the aziridinium cation 4b, which
is the active alkylating species (Scheme 2). Molecule 10
also indicates the possible structural variations that will
affect the conditions of the photochemical decaging
reaction. Electron-donating residues R′ and R′′ on the
aromatic ring are known to shift the absorption maxi-
mum of the photosensitive group further to the red, thus
allowing efficient irradiation with longer wavelengths.
On the other hand, residues R on the benzyl position are
known to affect the reaction rates and yields of the
decaging reaction. With these aspects in mind, it was
our goal to synthesize a number of structurally different
caged phosphamides and to compare their photochemical
behavior. In particular, we wanted to develop compounds
where R′ is not only an electron-donating residue but also
a linker that would allow attachment to other molecules.
These molecules include peptides and peptidomimetics
(18) Kwon, C.-H.; Moon, K.-Y.; Baturay, N.; Shirota, F. N. J . Med.
Chem. 1991, 34, 588-592.
(19) Friedman, O. M.; Seligman, A. M. J . Am. Chem. Soc. 1954, 76,
655-658.

2436 J . Org. Chem., Vol. 63, No. 8, 1998
Reinhard and Schmidt
Sch em e 4a
a
Reagents and conditions: (a) NaBH4, EtOH; (b) SnF2, C3H5I, DMF; (c) NaOH, BrCH2E, MeOH, reflux; (d) LHMDS, THF, 0 °C; 5;
then NH3(g); (e) 0.1 N NaOH (1 equiv), MeOH, 0 °C; (f) BH3‚SMe2 (excess), THF, rt; NaOH/H2O2; HCl.
moacetate to give 12a (R ) H) and 12b (R ) allyl) and
with bromoacetonitrile to yield 12c (R ) H) and 12d (R
) allyl). Sodium hydroxide was used as a base in both
cases. In the next step, we introduced the phosphor-
amide mustard moiety in the substituted 2-nitrobenzyl
alcohols. It turned out that the protected phosphoramide
mustards of this work, with the exeption of our model
compound 10a , could be prepared in a one pot synthesis
via direct reaction of phosphoric acid dichloride 5 and
the substituted 2-nitrobenzyl alcohols 8b,c and 12a -d
as outlined in Scheme 3. The alcohols 8b,c and 12a -d
were initially deprotonated with LHMDS, reacted with
the phosphoric acid dichloride 5, and finally converted
to the phosphoric acid diamidates 10b -g by bubbling
ammonia through the solution. Interestingly, in the case
of the nonsubstituted 2-nitrobenzyl alcohol 8a , this direct
route resulted in the phosphordiester amidate 15.
The carboxylic acid methyl ester functionalized com-
pounds 10d and 10e were converted into the correspond-
ing sodium carboxylates 13a and 13b by hydrolysis with
0.1 N aqueous sodium hydroxide. The nitrile 10g was
reduced to the amine by an excess of borane-dimethyl
sulfide complex; as the CdC of the allyl residue is also
hydroborated in this step as well, an intermediate
oxidation step of the alkylborane was necessary. The
final addition of hydrochloric acid yielded the ammonium
chloride salt 14.
hydrolyzable product was not expected to be a useful
prodrug with respect to their application in a biological
medium.
The products were characterized by 1H NMR, 13C NMR,
and 31P NMR spectra. The phosphorester diamidates
showed typical signals in their 31P NMR spectra. While
10a ,b,d ,f and 13a showed one singlet, the diastereomeric
mixtures of 10c,e,g and 14 appeared as two singlets in
the region between δ 22.54-21.15 ppm, indicating a
mixture of diasteromers. Attempts to separate any of the
diastereomers were not successful. The doubling of
signals in the carbon spectra reflects the appearance of
diasteromers as well. The formation of the phosphorester
bond in all “caged” phosphoramide mustards (10a -g and
13-15) was indicated by the 3J (P,C) coupling to the
benzylic carbon (primary, 64-66 ppm; secondary, 72-
3
75 ppm) in the range of 2.5-5.0 Hz and a J (P,C) coupling
in the range of 4.15-6.16 Hz to the R-carbon (48-50.5
ppm) of the bis(chloroethyl)amine moiety. Furthermore,
infrared spectra exhibited strong absorptions for the
carboxylic acid methyl ester groups at ν 1760 cm-1 for
10d and 10e and weak absorptions for the cyano groups
at ν 2260 and 2200 cm-1 for 10f and 10g, respectively.
The phosphoramidates 13-14 are highly water-soluble
compounds, while compounds 10a -g and 15 showed only
poor or no water solubility.
Even though the nitrile 10f could be reduced to its
amine with borane-dimethyl sulfide complex as well, the
product did not appear to be sufficiently stable toward
hydrolysis at the benzyl position for isolation of a pure
P h otolysis of th e Ca ged P h osp h or a m id e Mu s-
ta r d s. In the following section, the examination of the
photolysis properties of products 10a -g and 13-15 will
be described. The photochemical reaction of compound
10a (R, R′, R′′ ) H) is outlined in Scheme 2. As can be
seen from Figure 1, irradiation of a solution of the
nitrobenzyl phosphoramide mustard 10a in acetonitrile
(0.2 mM) for 20 s resulted in the expected changes in the
absorption spectrum. The nitrobenzyl chromophore of
1
compound. As shown by the H NMR spectrum of the
product mixture, there was always a significant amount
of the corresponding benzyl alcohol present (δ(CH2OH)
4.82 (s) ppm (D2O); δ(CH2OP) 5.25 (d) ppm (D2O)), even
after chromatographic purification. We did not make
further attempts to isolate this product, since an easily

Photosensitive Phosphoramide Mustards
J . Org. Chem., Vol. 63, No. 8, 1998 2437
F igu r e 1. UV spectral recording of the photolysis of nitroben-
zylphosphoramide mustard (10a ) in acetonitrile solution (0.2
mmol). The sample was photolyzed in a quartz cuvette (1 cm
path length) for 20 s to give 16 (λmax ) 304 nm).
10a showed an absorption maximum at λ 262 nm, the
nitrosobenzaldehyde photoproduct 16 (R, R′, R′′ ) H) at
λ 304 nm.
Since the absorption spectrum could only detect the
photochemical nitrosobenzaldehyde byproduct and not
the actual decaged substrate, we sought further proof of
the successful cleavage reaction. In this respect, 31P
NMR spectroscopy was a powerful tool. The conversion
of the phosphoric acid ester derivative 10a to the free
phosphoric acid derivative 4a (see Scheme 2) was ex-
pected to result in a significant upfield shift of the
observed 31P NMR resonance. As predicted, the free
phosphoric acid derivative 4a resonated at δ 3.15 ppm,
while the signal for the educt 10a was observed at δ 21.17
ppm (CD3CN/H2O (1:1)) (Figure 2). Solutions of higher
concentration lead to longer reaction times because of
internal filter effects of the nitrosobenzaldehyde photo-
product. The use of a 12 mM solution required 6 min
for an almost complete reaction in this experiment. Due
to further reactions of 4a in the aqueous medium (see
its metabolism in Scheme 1), a whole group of signals
was actually observed, which resulted in an overall broad
signal.
F igu r e 2. (a) 31P NMR spectrum of 10a in CD3CN/H2O
solution (1:1) (12 mM); (b) 31P NMR spectrum of the solution
in (a) after irradiation for 3 min in a 10 mm NMR tube (see
the Experimental Section for details).
The irradiation-coupled NBP assay was performed
with a mercury arc lamp with a maximum emission at λ
360 nm and a wavelength range of λ 300-400 nm. The
energy output of this lamp was measured to be 3.5% of
the EFOS mercury arc lamp used in the spectroscopic
photolysis studies (Figures 1 and 2). Since concentra-
tions of the irradiated sample solutions were much lower
for the NBP assay experiments than for the spectroscopic
studies (0.2 mM compared to 12 mM), the less powerful
illumination system yielded experimentally more conve-
nient time frames (0-10 min, see Figure 3). The use of
a filter system that transmitted light in the range λ 350-
500 nm with a maximum at λ 402 nm resulted in very
slow and incomplete reaction, even for the absorption red-
shifted derivative 10b. Therefore, all compounds were
photolyzed in the wavelength range of λ 300-400 nm.
It should be mentioned that, in all cases studied, there
was an absorption present at t ) 0 min (before irradia-
tion), which was slightly shifted to the blue (λ 515-520
nm) as compared to the chromophore for the alkylated
(nitrobenzyl)pyridine absorbing at λ 542 nm. To define
the origin of this absorption, we synthesized bis(2-
nitrobenzyl) phosphate (17) as a control (see the Experi-
mental Section) and performed the NBP assay with this
compound (t ) 0, 10 min). A weak absorption at λ 516
nm was observed and did not change during photolysis.
Other experiments showed that nonphosphorylated com-
pounds such as 12a do not have a similar absorption.
Therefore, we concluded that an adduct between the NBP
and the phosphate moiety21 is responsible for the “base-
line” absorption at λ ) 516 nm.
In addition to these spectroscopic investigations of the
cleavage reaction, an assay that elucidated the alkylating
activity of the irradiated solutions was desirable. It was
not only the goal to obtain a comparison of the overall
activity of the structurally different types of caged
phosphoramide mustards 10a -g and 13-15 but also to
determine the dependence of decaging rate on irradiation
time. Solutions were assayed for alkylating activity after
irradiation of the samples for 0, 2, 4, 6, and 10 min with
the well-established 4-(4-nitrobenzyl)pyridine (NBP) re-
agent.20 Prior to the interpretation of the resulting data
shown in Figure 3, some comments are necessary.
(20) We used a slightly modified procedure of the one described by:
Friedman, O. M.; Boger, E. Anal. Chem. 1961, 33, 906-910 (see
Experimental Section).
(21) Getz, M. E.; Watts, R. R. J . Assoc. Off. Agric. Chem. 1964, 47,
1094-1096.

2438 J . Org. Chem., Vol. 63, No. 8, 1998
Reinhard and Schmidt
at the benzyl position have been shown to stabilize the
aci-nitro intermediate of the photoreaction, thus enhanc-
ing the rate of photolysis.10
Comparison of the primary alcohol-derived phospho-
ramidates 10a ,b and 13a , with respect to their absorption
maxima, at (10a ) λmax 262 nm, (10b) λmax 346 nm, (13a )
λmax 316 nm, showed that irradiation with light of a
wavelength maximum at 360 nm was best suited for the
compound 10b. Although the rate of photorelease of 10a
was significantly slower, it reached the same overall
activity after 10 min as the 4,5-dimethoxy-substituted
2-nitrobenzyl derivative 10b. Compound 13a showed the
most efficient photorelease of the alkylating moiety of the
phosphoramides derived from primary benzylic alcohols.
In summary, we found a convenient method to syn-
thesize a number of nitrobenzyl-caged phosphoramide
mustards. This synthesis turned out to be a general
route, even if the compounds bear additional functional
groups. The main target molecules, the water-soluble
phosphoramide mustards 13a ,b and 14, were among the
most promising photosensitive alkylating agents. Pro-
drugs 13b and 14, based on secondary benzyl alcohol
caging moieties, exhibited the best results.
Our ongoing research on these compounds will focus
on the study of their antiproliferative effect on living cells.
Phosphoramide mustard, the active metabolite of cyclo-
phosphamide, is not stable as such. Our prodrugs are
well suited to the study of phosphoramide mustards in
different cell types in a time- and space-controlled
fashion, independent of metabolic prodrug transforma-
tions. Functional groups at the aromatic ring of the
caging moiety would allow linking these prodrugs to
molecules that mediate specific cellular uptake to inves-
tigate tissue specificity.
F igu r e 3. 4-(4-Nitrobenzyl)pyridine (NBP) assay of irradiated
solutions (t ) 0, 2, 4, 6, 10 min) of 10a -c and 15 (in CH3CN),
13a ,b and 14 (in H2O), and 17 (in EtOH/H2O 1:1). The relative
alkylating activity was determined by measuring the absor-
bance of the alkylated NBP chromophore at λ ) 542 nm. The
numbers are relative numbers. See the text and Experimental
Section for details.
In addition to the baseline absorption, there are weak
absorptions present at λ 542 nm for some compounds
even without irradiation. Typically, the derivatives of
the primary nitrobenzyl group exhibited this phenom-
enon, which probably originated from hydrolysis of the
compounds due to the rather harsh reaction conditions
(boiling in an aqueous buffer solution, pH 4.6, for 20 min)
required for the NBP assay. Bis(nitrobenzyl)phosphora-
mide mustard 15 showed the highest final alkylation
activity. The compound’s final absorption (t ) 10 min)
at its maximum at λ 542 nm was set to 1, and all other
absorption values were normalized. The phosphordiester
amidate 15 also showed the highest baseline absorption.
This compound was more likely to undergo hydrolysis
under NBP assay conditions than the phosphorester bis-
(amidates), thus showing a higher baseline value. The
data presented in Figure 3 were finally corrected for their
baseline absorption to take this nonillumination-based
effect into account. When the irradiation was continued
after the compounds exhibited their maximum activity,
the curves tended to drop slightly, indicating reactions
of the photolyzed reaction products. The results in
Figure 3 can be summarized as follows:
Exp er im en ta l Section
Syn th esis. Gen er a l Meth od s. All reactions were con-
ducted under a dry argon atmosphere and under subdued
light. Anhydrous THF (Aldrich Chem. Co.) and all reagents
were used as received. Other solvents were reagent grade and
used without further purification. 1H NMR spectra were
recorded at 300 MHz with the residue solvent peak used as
reference relative to TMS; 31P NMR spectra were run at 121
MHz with trimethyl phosphite (1% in benzene) as external
standard (δ 141 ppm). Flash chromatography was performed
on silica gel (J . T. Baker, 40 µm particle size). Melting points
are not corrected. Elemental analyses were performed by
Atlantic Microlab, Inc. (Norcross, GA).
Sa fety Note. Because cyclophosphamide is a potent bio-
logical alkylating agent, it is only prudent to assume that
derivatives of its active metabolite phosphoramide mustard
are also potentially toxic and carcinogenic. At all times,
efficient hoods and protective clothing should be used in
working with these substances.
Bis(2-ch lor oeth yl)p h osp h or a m id ic d ich lor id e (5) was
prepared as previously described.19
2-Nitr oben zyl N,N-Bis(2-ch lor oeth yl)p h osp h or d ia m i-
d a te (10a ). Bis(2-chloroethyl)phosphoramidic dichloride 5 (0.2
g, 0.77 mmol) was dissolved in 5 mL of THF, the solution cooled
to 0 °C, and hexamethyldisilazane (0.33 mL, 1.56 mmol) was
slowly added. The solution was brought to room temperature
and stirred for 15 h. A white precipitate was formed. Ether
(15 mL) was added, and the flask was put in a freezer (-22
°C) for 1 h. The mixture was filtered, and the solvents were
removed. The resulting colorless oil was dissolved in THF (10
mL). Meanwhile, 2-nitrobenzyl alcohol (8a ) (0.12 g, 0.78 mmol)
was dissolved in 10 mL of THF and cooled to 0 °C, and a 1 M
solution of LHMDS in THF (0.78 mL, 0.78 mmol) was slowly
added. This solution was stirred at 0 °C for 10 min and then
Bis(nitrobenzyl)phosphoramide mustard 15 cleaved
with the slowest reaction of all compounds. The il-
lumination-mediated final alkylating activity of 15 did
not significantly differ from 10d , 13b, and 14, considering
that its t ) 0 value was higher than the other derivatives.
The derivatives 13b and 10c with an allyl substitutent
at the benzyl position exhibited faster photochemical
conversions than their nonsubstituted analogues 13a and
10a . The maximum activity for 13b, 10c, and 14 was
reached within the first 2 min of irradiation, with 13b
exhibiting the highest alkylating activity. Substituents

Photosensitive Phosphoramide Mustards
J . Org. Chem., Vol. 63, No. 8, 1998 2439
added with vigorous stirring at 0 °C to the above phosphor-
diamide solution by means of a transfer needle. The resulting
yellow solution was kept at 0 °C for 3 h. The THF was
evaporated, and the resulting oil was extracted with CH2Cl2
(50 mL) and water (50 mL). The layers were separated, and
the aqueous layer was extracted (2×) with 20 mL of CH2Cl2.
After evaporation of the solvent, an oil resulted, which
primarily consisted of 8a and product 10a . Using flash
chromatography, 8a was separated by elution with EtOAc.
During chromatography, the silyl groups of the phosphordia-
midate were rapidly hydrolyzed. Product 10a was eluted with
a 5:1 mixture of EtOAc/MeOH (Rf 0.55) and crystallized from
ether to yield 0.18 g (66%) of yellow crystals: mp 76 °C; IR
solvent (elutes mainly 8c), followed by EtOAc (Rf 0.5), which
gave 10c as a yellow oil: yield 41%; IR (film) 1255 (s, PdO)
cm-1; 1H NMR (CD3CN) δ 7.94 (d, J ) 8.7 Hz, 1 H), 7.78-7.68
(m, 2 H), 7.51 (m, 1 H), 5.93-5.81 (m, 2 H), 5.11-5.05 (m, 2
H), 3.62 (t, J ) 7.2 Hz, 2 H), 3.53-3.03 (m, 6 H), 2.71-2.57
(m, 2 H); 13C NMR (acetone-d6) δ 148.4/148.2, 137.9/137.6,
134.3/132.2, 134.2, 129.6/129.5, 129.4, 125.0, 118.6, 72.7 (J CP
) 3.65 Hz)/72.6 (J CP ) 3.68 Hz), 50.2, 43.1, 42.9/42.8; 31P NMR
(CD3CN) δ 21.54, 21.50 (2s); UV (CH3CN) λmax 260 nm (ꢀmax
4760). Anal. Calcd for C14H20Cl2N3O4P: C, 42.44; H, 5.09;
N, 10.61. Found: C, 42.71; H, 5.13; N, 10.50.
4-Hydr oxy-2-(h ydr oxym eth yl)-1-n itr oben zen e (11a) was
prepared by dissolving 5-hydroxy-2-nitrobenzaldehyde (7c)
(8.45 g, 50 mmol) in 50 mL of 1 N sodium hydroxide solution
(1 equiv) and adding 0.95 g (25 mmol) of sodium borohydride
pellets. This solution was stirred for 4 h, acidified to pH 2
with 1 N HCl, and extracted with EtOAc (4 × 50 mL).
Removal of the solvent yielded a pale yellow powder: mp 105
°C; yield 93%; IR (KBr): 3400 (m, OH), 3100 (s, br, OH), 1540
1
(KBr) 1260 (s, PdO) cm-1; H NMR (CD3CN) δ 8.09 (dd, J )
8.0, 0.9 Hz, 1 H), 7.81 (d, J ) 8.0 Hz, 1 H), 7.74 (dt, J ) 8.0,
1.0 Hz, 1 H), 7.54 (t, J ) 8.0 Hz, 1 H), 5.30 (d, J ) 6.9 Hz, 2
H), 3.63 (t, J ) 6.9 Hz, 4 H), 3.46-3.42 (br, 2 H), 3.46-3.22
(m, 4 H);13C NMR (acetone-d6) δ 147.8, 134.7, 134.6, 134.4,
129.3, 125.3, 63.9 (J CP ) 2.57 Hz), 50.1 (J CP ) 4.23 Hz), 43.1;
31P NMR (CD3CN) δ 22.00; UV (CH3CN) λmax 262 nm (ꢀmax
5520). Anal. Calcd for C11H16Cl2N3O4P: C, 37.09; H, 4.53;
N, 11.80. Found: C, 37.24; H, 4.56; N, 11.74.
1
(s, NO2) cm-1; H NMR (DMSO-d6) δ 10.91 (s, br, 1 H), 8.05
(d, J ) 9.0 Hz, 1 H), 7.24 (d, J ) 2.8 Hz, 1 H), 6.78 (dd, J )
9.0, 2.8 Hz, 1 H), 5.52 (t, J ) 5.4 Hz, 1 H), 4.80 (d, J ) 5.4 Hz,
2 H); 13C NMR (acetone-d6)δ 163.6, 143.3, 140.3, 128.4, 115.3,
114.8, 62.0.
4,5-Dim eth oxy-2-n itr oben zyl Alcoh ol (8b). This com-
pound was prepared by sodium borohydride reduction of
6-nitroveratraldehyde (7a ) in ethanol solution: yield 96%; mp
4-H yd r oxy-2-(1-h yd r oxy-3-b u t e n yl)-1-n it r ob e n ze n e
(11b). This compound was prepared analogously to 8c from
2.00 g (12.0 mmol) of 5-hydroxy-2-nitrobenzaldehyde (7c), 1.10
mL (1 equiv) of allyl iodide, and 1.88 g (1 equiv) of stannous
fluoride: yield 94% of a pale yellow oil; IR (KBr) 3500 (w, OH),
1
142 °C; IR (KBr) 3500 (s, OH), 1520 (s, NO2) cm-1; H NMR
(CDCl3) δ 7.59 (s, 1 H), 7.07 (s, 1 H), 4.86 (d, J ) 6.5 Hz, 2 H),
3.90 (s, 3 H), 3.85 (s, 3 H), 2.64 (t, J ) 6.5 Hz, 1 H); 13C NMR
(acetone-d6) δ 154.9, 148.4, 139.7, 134.8, 110.4, 108.6, 61.7,
56.4.
1
3280 (s, br, OH), 1510 (s, NO2) cm-1; H NMR (CDCl3) δ 7.97
1-(2-Nit r op h en yl)-3-bu t en -1-ol (8c). 2-Nitrobenzalde-
hyde (7b) (2.00 g, 13.23 mmol) was dissolved in DMF (30 mL),
and 1.25 mL (1 equiv) of allyl iodide was added. The solution
was cooled to 0 °C, and stannous fluoride (2.07 g, 1 equiv) was
added. The ice bath was removed, and the solution was stirred
for 2 h. A slightly exothermic reaction occurred. Water (60
mL) was poured into the DMF solution, and the aqueous layer
was extracted (3×) with 30 mL of ether. The organic layer
was then washed with 2 × 50 mL of saturated NH4Cl solution.
After evaporation of the ether, a yellow oil remained; its
identity as 8c was checked by 1H NMR, and it was used
without further purification: yield 74%; IR (film) 3400 (br, m,
(d, J ) 8.8 Hz, 1 H), 7.20 (d, J ) 2.5 Hz, 1 H), 6.97 (s, br, 1 H),
6.74 (dd, J ) 8.8, 2.5 Hz, 1 H), 5.91-5.77 (m, 1 H), 5.46 (dd,
J ) 8.2, 3.6 Hz, 1 H), 5.08-5.01 (m, 2 H), 2.80-2.40 (s, br, 1
H), 2.70-2.62 (m, 1 H), 2.29 (m, 1 H); 13C NMR (CDCl3) δ
161.1, 142.6, 140.2, 133.6, 128.2, 119.1, 115.1, 114.2, 69.3, 42.3.
Meth yl [3-(Hyd r oxym eth yl)-4-n itr oph en oxy]eth a n oa te
(12a ). Typ ica l P r oced u r e. Phenol 11a (3.25 g, 19.0 mmol)
was placed in a flask and dissolved in MeOH (50 mL). NaOH
pellets (0.76 g, 1 equiv) were powdered and added to the
solution. The color of the solution turned deep yellow as the
NaOH gradually dissolved. The solution was kept at room
temperature for 15 h. Methyl bromoacetate (1.98 mL, 1.1
equiv) was added, and the mixture was heated to reflux (oil
bath temperature: 100 °C) for 15 h. After cooling, the MeOH
was evaporated and the solid residue dissolved in CH2Cl2 (50
mL) and water (100 mL), which was brought to pH 9 by adding
solid K2CO3. Extraction was repeated twice with 50 mL of
CH2Cl2. The combined organic layer was washed (3×) with
30 mL of water. After evaporation of the solvent, the remain-
ing solid was recrystallized from chloroform-ether to yield 3.26
g (71%) of 12a as a pale yellow powder: mp 120 °C; IR (KBr)
1
OH), 3080 (w, dCH), 1525 (vs, NO2) cm-1; H NMR (CD3CN)
δ 7.85 (dd, J ) 8.0, 1.2 Hz, 1 H), 7.79 (dd, J ) 7.9, 1.2 Hz, 1
H), 7.66 (dt, J ) 8.0, 1.2 Hz, 1 H), 7.43 (dt, J ) 7.7, 1.5 Hz, 1
H), 5.96-5.82 (m, 1 H), 5.22-5.17 (m, 1 H), 5.10-5.02 (m, 2
H), 3.57 (d, br, J ) 3.6 Hz, 1 H), 2.57-2.49/2.44-2.34 (m, 2
H); 13C NMR (acetone-d6) δ 148.5, 141.0, 135.5, 133.7, 129.0,
128.6, 124.4, 117.5, 68.9, 43.7.
4,5-Dim eth oxy-2-n itr oben zyl N,N-Bis(2-ch lor oeth yl)-
p h osp h or d ia m id a te (10b). Typ ica l P r oced u r e. Alcohol
8b (0.60 g, 2.81 mmol) was dissolved in 20 mL of THF and
cooled to 0 °C, and a solution of LHMDS in THF (1 M, 2.8
mL, 2.8 mmol) was slowly added. This mixture was stirred
at 0 °C for 10 min. Meanwhile, 5 (0.72 g, 2.80 mmol) was
dissolved in 20 mL of THF and cooled to 0 °C. The solution of
the alkoxide was transferred into this flask by means of a
cannula, and stirring was continued for 1 h at 0 °C. Ammonia
was bubbled through the solution at a moderate rate for 30
min at 0 °C, and then the solution was warmed to room
temperature and stirred for an additional 2 h. The same
workup procedure was applied as described for compound 10a ,
Rf (EtOAc/MeOH 5:1) 0.50. The product formed a pale yellow
powder when ether was added: mp 121 °C; yield 60%; IR (KBr)
1
1740 (vs, CdO) cm-1; H NMR (CDCl3) δ 8.12 (d, J ) 9.1 Hz,
1 H), 7.20 (d, J ) 3.0 Hz, 1 H), 6.83 (dd, J ) 9.2, 2.9 Hz, 1 H),
4.94 (d, J ) 4.4 Hz, 2 H), 4.69 (s, 2 H), 3.76 (s, 3 H); 13C NMR
(acetone-d6) δ 169.7, 163.3, 142.9, 141.6, 128.1, 114.7, 113.8,
66.0, 61.9, 52.4.
Met h yl [3-(1-H yd r oxy-3-b u t en yl)-4-n it r op h en yloxy]-
eth a n oa te (12b). This compound was prepared using the
procedure described for 12a to give 65% of 12b as yellow
crystals; crystallization was achieved from ether-hexane: mp
64 °C; IR (KBr) 1770 (s, CdO) cm-1; 1H NMR (CD3CN) δ 8.00
(d, J ) 9.4 Hz, 1 H), 7.27 (d, J ) 2.7 Hz, 1 H), 6.91 (dd, J )
9.4, 2.7 Hz, 1 H), 5.98-5.84 (m, 1 H), 5.33 (quintet, J ) 4.1
Hz, 1 H), 5.09-5.03 (m, 2 H), 4.80 (s, 2 H), 3.74 (s, 3 H), 3.51
(d, J ) 4.6 Hz, 1 H); 13C NMR (acetone-d6) δ 169.2, 162.8, 145.2,
142.2, 135.8, 127.7, 117.5, 114.7, 114.3, 69.3, 66.0, 52.4, 43.8.
[3-(Hydr oxym eth yl)-4-n itr oph en oxy]eth an en itr ile (12c).
This compound was prepared analogously to 12a using bro-
moacetonitrile to give 66% of 12c as a pale brown powder that
was crystallized from ether: mp 103 °C; IR (KBr): 3500 (vs,
br, OH), 1510 (vs, NO2) cm-1; 1H NMR (CD3CN) δ 8.17 (d, J )
9.0 Hz, 1 H), 7.45 (d, J ) 2.9 Hz, 1 H), 7.04 (dd, J ) 9.1, 2.8
Hz, 1 H), 5.01 (s, 2 H), 4.94 (s, 2 H), 3.59 (br, 1 H); 13C NMR
(acetone-d6) 161.7, 143.1, 142.5, 128.2, 116.0, 114.8, 114.1, 61.7,
54.7.
1
1280 (vs, PdO) cm-1; H NMR (CD3CN) δ 7.67 (s, 1 H), 7.21
(s, 1 H), 5.42 (dd, J ) 7.0, 3.9 Hz, 2 H), 3.87 (s, 6 H), 3.68 (t,
J ) 6.7 Hz, 4 H), 3.45 (dt, J ) 11.7, 6.7 Hz, 4 H); 13C NMR
(acetone-d6) δ 155.1, 149.4, 140.4, 129.7 (J CP ) 8.4 Hz), 111.5,
109.3, 64.3 (J CP ) 3.3 Hz), 56.9, 56.8, 50.5 (J CP ) 4.4 Hz), 43.3;
31P NMR (CD3CN) δ 21.90; UV (CH3CN) λmax 346 nm (ꢀmax
6875). Anal. Calcd for C13H20Cl2N3O6P: C, 37.51; H, 4.84;
N, 10.10. Found: C, 37.51; H, 4.85; N, 10.00.
3-Bu t en -1-(2-n it r op h en yl) 1-N,N-b is(2-ch lor oet h yl)-
p h osp h or d ia m id a te (10c) was prepared as described above
for 10b. For flash chromatography, ether was used as the first

2440 J . Org. Chem., Vol. 63, No. 8, 1998
Reinhard and Schmidt
[3-(1-H yd r oxy-3-b u t en yl)-4-n it r op h en oxy]et h a n en i-
tr ile (12d ). This compound was prepared analogously to 12a
using bromoacetonitrile to give 84% of 12d as a yellow oil. The
oil was further purified by column chromatography on silica
gel (eluent: hexane/ether 1:1) to give a yellow solid: mp 47-
Hz, 1 H), 7.11-7.06 (m, 1 H), 6.04-5.97 (m, 1 H), 5.96-5.81
(m, 1 H), 5.11-5.04 (m, 2 H), 5.03/5.01 (s, 2 H), 3.66-3.12 (m,
8 H), 2.75-2.53 (m, 2 H); 13C NMR (acetone-d6) δ 161.3, 142.9/
142.8, 141.7/141.2 (J CP ) 3.16 Hz), 134.2/134.1, 128.2, 118.8,
116.1, 115.9/115.8, 115.4/115.2, 72.9 (J CP ) 4.2 Hz), 72.8 (J CP
) 4.2 Hz), 54.9/54.8, 50.5 (J CP ) 4.8 Hz)/50.4 (J CP ) 4.5 Hz),
43.3/43.2, 42.8 (J CP ) 4.7 Hz)/42.7 (J CP ) 5.0 Hz); 31P NMR
1
49 °C; IR (film) 2250 (w, CN) cm-1; H NMR (CD3CN) δ 8.03
(d, J ) 8.8 Hz, 1 H), 7.40 (d, J ) 3.0 Hz, 1 H), 7.02 (dd, J )
8.8, 3.0 Hz, 1 H), 5.98-5.85 (m, 1 H), 5.34 (m, 1 H), 5.13-5.04
(m, 2 H), 5.00 (s, 2 H), 3.60 (d, J ) 4.5 Hz, 1 H), 2.58-2.49 (m,
1 H), 2.34 (m, 1 H); 13C NMR (acetone-d6) δ 161.0, 145.1, 142.8,
135.5, 127.8, 117.6, 115.9, 114.7, 114.5, 69.1, 54.5, 43.6. Anal.
Calcd for C12H12N2NO4: C, 58.06; H, 3.4.87; N, 11.29. Found:
C, 58.21; H, 4.93; N, 11.24.
(CD3CN) δ 21.52, 21.41 (2 s). Anal. Calcd for C16H21
-
Cl2N4O5P: C, 42.59; H, 4.69; N, 12.42. Found: C, 42.41; H,
4.78; N, 12.27.
[3-Meth yl-N,N-bis(2-ch lor oeth yl)p h osp h or d ia m id a to-
4-(n itr op h en yl)oxy]a ceta te, Sod iu m Sa lt (13a ). Typ ica l
P r oced u r e. Methyl ester 10d (0.14 g, 0.315 mmol) was
dissolved in 10 mL of MeOH and cooled to 0 °C, and 3.15 mL
of 0.1 N NaOH solution (3.15 mmol) was added dropwise. The
solution was stirred at room temperature for 3 h. Most of the
solvent was evaporated, and 2-propanol was added until the
solution became cloudy. The flask was put in a freezer (-22
°C); the product was obtained as a pale yellow powder: mp
145 °C; yield 63%; IR (KBr) 1620 (vs, CdO), 1285 (s, PdO)
5-[(Ca r bom eth oxy)m eth oxy]-2-n itr oben zyl N,N-Bis(2-
ch lor oeth yl)p h osp h or d ia m id a te (10d ). Typ ica l P r oce-
d u r e. Methyl [3-(hydroxymethyl)-4-nitrophenoxy]ethanoate
(12a ) (0.2 g, 0.83 mmol) was dissolved in THF (20 mL) and
cooled to 0 °C. A 1 M solution of LHMDS in THF (0.91 mL,
1.1 equiv) was slowly added with vigorous stirring, and the
solution was kept at 0 °C for 10 min. Meanwhile, 5 (0.24 g,
1.1 equiv) was dissolved in 20 mL of THF and cooled to 0 °C.
The solution of the alkoxide was transferred into this flask by
means of a transfer needle, and stirring was continued for 1 h
at 0 °C. Ammonia was bubbled through the solution at a
moderate rate for 30 min at 0 °C, and stirring was continued
for 30 min at that temperature. The THF was evaporated
without heating the solution. The oily residue was dissolved
in 50 mL of CH2Cl2 and 50 mL of water, and the aqueous layer
was extracted twice with 25 mL of CH2Cl2. The resulting
almost colorless oil was purified by flash chromatography.
Initial elution with EtOAc separated byproducts. The product
was eluted with EtOAc/MeOH (5:1) (Rf 0.4) and crystallized
from CH2Cl2-ether to yield 0.15 g (40%) of 10d as pale yellow
crystals: mp 109 °C; IR (KBr) 1760 (vs, CdO), 1230 (vs, PdO)
cm-1; 1H NMR (CD3CN) δ 8.15 (d, J ) 9.1 Hz, 1 H), 7.28 (d, J
) 2.8 Hz, 1 H), 6.95 (dd, J ) 9.1, 2.8 Hz, 1 H), 5.30 (dd, J )
6.5, 2.8 Hz, 2 H), 4.81 (s, 2 H), 3.74 (s, 3H), 3.64 (t, J ) 7.4
Hz, 4 H), 3.58-3.34 (m, 4 H); 13C NMR (CD3CN) δ 169.6, 163.4,
141.8, 138.1 (J CP ) 8.0 Hz), 128.8, 115.3, 114.7, 66.5, 64.6 (J CP
) 3.2 Hz), 53.0, 50.2 (J CP ) 4.4 Hz), 43.6; 31P NMR (CD3CN)
δ 22.27 (s).
1
cm-1; H NMR (D2O) δ 7.99 (d, J ) 9.3 Hz, 1 H), 7.00 (d, J )
2.8 Hz, 1 H), 6.77 (dd, J ) 9.3, 2.8 Hz, 1 H), 5.16 (d, J ) 6.6
Hz, 2 H), 4.41 (s, 2 H), 3.53 (t, J ) 6.4 Hz, 4 H), 3.30 (dt, J )
11.1, 6.4 Hz, 4 H), 3.17 (s, 2 H); 13C NMR (CD3OD/D2O)δ 174.5,
164.5, 141.0, 137.4 (J CP ) 8.7 Hz), 128.7, 115.3, 115.1, 68.4,
65.2 (J CP ) 3.3 Hz), 50.2 (J CP ) 4.5 Hz), 43.3 (J CP ) 1.4 Hz);
31P NMR (D2O) δ 22.41 (s); UV (H2O) λmax 316 nm (ꢀmax 9460).
Anal. Calcd for C13H17Cl2N3NaO7P: C, 34.53; H, 3.79; N, 9.29.
Found: C, 34.65; H, 3.86; N, 9.17.
3-[3-Bu ten yl-1-(N,N-bis(2-ch lor oeth yl)p h osp h or d ia m i-
d a to)]-4-(n itr op h en yl)oxya ceta te, Sod iu m Sa lt (13b). This
compound was prepared from methyl ester 10e using the
procedure given for 13a to give 70% of 13b as a brown
powder: mp 120 °C; IR (KBr) 1615 (vs, CdO), 1285 (s, PdO)
1
cm-1; H NMR (D2O) δ 8.00/7.99 (2 d, J ) 9.4 Hz, 1 H), 7.07/
7.04 (2 d, J ) 2.7 Hz, 1 H), 6.84 (m, 1 H), 5.96-5.89 (m, 1 H),
5.85-5.68 (m, 1 H), 5.01-4.94 (m, 2 H), 4.45 (s, 2 H), 3.53/
3.34 (2 t, J ) 6.6 Hz, 4 H), 3.43-2.97 (m, 4 H), 2.55/2.50 (2 m,
2 H); 13C NMR (D2O) δ 175.2/175.0, 162.8, 140.1/139.5, 139.7/
139.6, 133.2/133.0, 128.1/128.0, 119.1, 114.9/114.4, 113.7, 73.2
(J CP ) 3.67 Hz)/72.8 (J CP ) 3.67 Hz), 67.3/67.2, 48.1 (J CP
)
3-Bu t e n yl-1-[5-[(Ca r b om e t h oxy)m e t h oxy]-2-n it r o-
p h en yl] 1-N,N-b is(2-ch lor oet h yl)p h osp h or od ia m id a t e
(10e). This compound was prepared from alcohol 12b accord-
ing to the procedure described for 10d to give 42% of 10e as a
mixture of diastereoisomers: colorless oil; Rf (EtOAc) 0.4; IR
4.15 Hz)/47.9 (J CP ) 4.31 Hz), 42.3/42.2, 41.7/41.6; 31P NMR
(D2O) δ 21.09 (s); UV (H2O) λmax 316 nm (ꢀmax 8170). Anal.
Calcd for C16H21Cl2N3NaO7P: C, 39.04; H, 4.30; N, 8.54.
Found: C, 38.98; H, 4.37; N, 8.45.
(film) 1760 (s, CdO), 1250-1200 (vs, PdO) cm-1 1H NMR
;
[3-(Bu t a n -4-ol-1-(N,N-b is(2-ch lor oet h yl)p h osp h or d i-
a m id a t o)-4-n it r op h en yl)oxy]-2-et h yla m m on iu m Ch lo-
r id e (14). The nitrile 10g (0.35 g, 0.77 mmol) was dissolved
in THF, and a 2 M solution of borane-dimethyl sulfide
complex in THF (2.31 mL, 6 equiv) was added dropwise at
room temperature. Evolution of gas occurred for about 5 min.
The solution was stirred for 15 h at room temperature. The
flask was placed in a water bath (20 °C), and 4.62 mL (4.62
mmol, 6 equiv) of a 1 N solution of NaOH was added, followed
by 0.48 mL (4.62 mmol, 6 equiv) of a 30% solution of H2O2.
This mixture was stirred for 2 h at room temperature;
eventually, a white solid separated. After filtration and
removal of the solvents, water and dilute HCl were added to
bring the pH to 1. The aqueous layer was extracted twice with
30 mL of EtOAc and then brought to pH 9 by addition of a
K2CO3 solution, and the product was extracted with EtOAc (4
× 40 mL). The solvent was evaporated, the remaining oil
weighed and dissolved in MeOH (10 mL), and a slight excess
of the theoretical amount of 0.1 N HCl was added. Solvents
were removed, the oily residue dissolved in EtOH, and ether
was added until the solution became cloudy. On standing at
-22 °C, the product was obtained as a yellow powder: mp 115
°C; yield 54%; 1H NMR (D2O) δ 8.06 (d, J ) 9.1 Hz, 1 H), 7.21
(d, J ) 2.8 Hz, 1 H), 6.99 (dd, J ) 9.1, 2.8 Hz, 1 H), 5.93 (m,
1 H), 4.32 (t, J ) 4.9 Hz, 2 H), 3.58 (t, J ) 6.6 Hz, 4 H), 3.51
(m, 2 H), 3.37 (t, br, J ) 4.7 Hz, 2 H), 3.35-3.28 (m, 4 H),
1.90-1.83 (m, 2 H), 1.62-1.59 (m, 2 H); 13C NMR (D2O) δ
162.7, 140.8, 140.5, 128.6, 114.7, 114.13, 75.0 (J CP ) 5.1 Hz),
65.0, 61.6, 48.1 (J CP ) 5.0 Hz), 42.4, 41.39.2, 34.1 (J CP ) 6.5
(CD3CN) δ 8.04 (d, J ) 9.4 Hz, 1 H), 7.21/ 7.18 (2d, J ) 3.0
Hz, 1 H), 6.97 (dd, J ) 9.4, 3.0 Hz, 1 H), 6.03-5.96 (m, 1 H),
5.95-5.81 (m, 1 H), 5.10-5.04 (m, 2 H), 4.82, 4.81 (2s, 2 H),
3.75 (s, 3 H), 3.63 (t, J ) 7.4 Hz, 1 H), 3.54-3.05 (m, 7 H),
2.73-2.53 (m, 2 H); 13C NMR (CD3CN) δ 169.6/169.5, 162.9/
162.8, 142.1, 141.5/141.1, 134.5/134.4, 128.4, 119.2/119.1,
115.5/115.4, 115.2/115.1, 73.1 (J CP ) 4.36 Hz)/73.0 (J CP ) 4.43
Hz), 66.5, 53.0, 50.2 (J CP ) 4.65 Hz)/50.1 (J CP ) 6.16 Hz), 43.6
(J CP ) 2.78)/43.4 (J CP ) 2.78); 31P NMR (CD3CN) δ 21.32, 21.15
(2 s).
5-(Cya n om et h oxy)-2-(n it r ob en zyl) N,N-Bis(2-ch lor o-
eth yl)p h osp h or od ia m id a te (10f). This compound was pre-
pared from alcohol 12c according to the procedure described
for 10d to give 44% of 10f as an orange oil: Rf (EtOAc/MeOH
5:1) 0.5; IR (film) 2260 (w, CN), 1250-1200 (s, PdO) cm-1; 1H
NMR (CD3CN) δ 8.19 (d, J ) 8.9 Hz, 1 H), 7.37 (d, J ) 2.8 Hz,
1 H), 7.08 (dd, J ) 9.0, 2.9 Hz, 1 H), 5.34 (d, J ) 6.0 Hz, 2 H),
5.03 (s, 2 H), 3.65 (t, J ) 6.8 Hz, 4 H), 3.45-3.37 (m, 4 H); 13
C
NMR (CD3CN) δ 161.9, 142.7, 138.1 (J CP ) 8.55 Hz), 128.9,
116.2, 115.8, 115.1, 64.7 (J CP ) 3.4 Hz), 61.1, 55.4, 50.2 (J CP
)
4.5 Hz), 43.6; 31P NMR (CD3CN) δ 22.52 (s).
3-Bu ten yl-1-[5-(cya n om eth oxy)-2-n itr op h en yl] 1-N,N-
Bis(2-ch lor oeth yl)p h osp h or d ia m id a te (10g). This com-
pound was prepared from alcohol 12d according to the
procedure described for 10d to give 52% of 10g as a mixture
of two diastereoisomers: yellow oil; Rf (EtOAc/MeOH 5:1) 0.55;
IR (film) 2200 (w, CN), 1285 (s, PdO) cm-1 1H NMR (CD3-
;
CN) δ 8.09, 8.08 (2 d, J ) 8.9 Hz, 1 H), 7.30, 7.29 (2 d, J ) 3.0

Photosensitive Phosphoramide Mustards
J . Org. Chem., Vol. 63, No. 8, 1998 2441
Hz), 28.0; 31P NMR (D2O) δ 21.70/ 21.32 (2 s); UV (H2O) λmax
308 nm (ꢀmax 7350).
uct and the 31P NMR spectroscopic detection of released
phosphoramide mustard were performed with a mercury arc
lamp (EFOS Ultracure 100ss plus; output 1 W/cm2). The
irradiations in connection with the NBP assay were performed
with a mercury arc lamp (Carl Zeiss, HBO 200W/4, L1; output
35 mW/cm2). The output was passed through a glass filter
that transmitted light of wavelengths λ 300-400 nm (λmax 362
nm). The samples were mounted at a 2 cm distance from the
optics and were cooled during photolysis by a fan.
Ir r a d ia tion s for Assa yin g Alk yla tin g Activity. Pho-
tolysis was performed in quartz cuvettes (1 cm path length);
3 mL of 0.2 mM solutions, nonstirred, were used: 10a -c and
15 in CH3CN, 13a ,b and 14 in H2O, 17 in EtOH/H2O (1:1).
Aliquots were taken after t ) 0 (without irradiation), 2, 4, 6,
and 10 min and used in the following procedure.
Det er m in a t ion of t h e Alk yla t in g Act ivit y b y NBP
Assa y. The known procedure20 was slightly modified. In each
of five screw-capped and round-bottomed 10 mL test tubes
were placed 2 mL of distilled water, 1 mL of 0.025 M NaOAc
buffer (pH 4.6), and 0.5 mL of 5% NBP in acetone. Two
hundred µL aliquots of the irradiated sample (t ) 0, 2, 4, 6,
and 10 min) were added and the test tubes placed in a boiling
water bath for 20 min. The samples were then immediately
placed in an ice bath. One mL of acetone and 3 mL of ethyl
acetate were added to each tube. Handling one tube at a time,
1 mL of a 0.25 N NaOH solution was added and the tube
immediately vortexed for 30 s; 1 min was allowed for complete
phase separation, and then a sample (1.5 mL) of the upper
phase was transferred into a quartz cuvette and the absorption
measured at λ 542 nm. Because the baseline of the five
different readings tended to shift slightly, a baseline absorption
at λ 700 nm was also read and subtracted from the chro-
mophore absorption. The reading with the overall maximum
absorption (compound 10a after t ) 10 min) was set to 1.00,
and all other readings were scaled in relation to this number.
For the illustration of the illumination-based NBP absortion
in Figure 3, the t ) 0 absorption value was subtracted: 0.234
(10a ), 0.0990 (10b), 0.190 (10c), 0.260 (15), 0.0790 (13a ),
0.0500 (13b), 0.112 (14), 0.134 00 (17)).
Bis(2-n it r ob en zyl) N,N-Bis(2-ch lor oet h yl)p h osp h or -
a m id a te (15). Alcohol 8a (0.6 g, 3.92 mmol) was dissolved in
THF (15 mL), and the solution was cooled to 0 °C. A 1 M
solution of LHMDS in THF (3.9 mL, 1 equiv) was slowly added,
and stirring was continued for 10 min at 0 °C. Bis(2-
chloroethyl)phosphoramidic dichloride (5) (0.51 g, 1.96 mmol)
was added in small portions, and the solution was kept at 0
°C for 45 min. The THF was evaporated, and the oily residue
was extracted with CH2Cl2 (3 × 3 0 mL) and water. The
resulting oil was subjected to flash chromatography. The first
fraction, eluted with ether/CH2Cl2, contained mainly 8a .
Product 15 eluted cleanly with EtOAc as solvent (Rf 0.7) and
was crystallized as colorless crystals from ether/hexane: mp
81 °C; yield 47%; IR (KBr): 1260 (s, PdO) cm-1; 1H NMR (CD3-
CN) δ 8.09 (d, J ) 8.0 Hz, 2 H), 7.73 (m, 4 H), 7.56 (m, 2 H),
5.43 (d, J ) 7.0 Hz, 4 H), 3.65 (t, J ) 6.8 Hz, 4 H), 3.42 (dt, J
) 11.5, 6.7 Hz, 4 H); 13C NMR (acetone-d6) δ 148.4, 134.8, 133.3
(J CP ) 7.93 Hz), 130.0, 129.8, 125.6, 65.6 (J CP ) 3.99 Hz), 49.8
(J CP ) 4.28 Hz), 42.8; 31P NMR (CD3CN) δ 14.78 (s); UV (CH3-
CN) λmax 262 nm (ꢀmax 9330). Anal. Calcd for C18H20
-
Cl2N3O7P: C, 43.92; H, 4.10; N, 8.54. Found: C, 44.02; H,
4.06; N, 8.51.
Bis-2-n itr oben zyl P h osp h a te (17).22 Methyl dichloro-
phosphate (0.1 mL, 0.98 mmol) and 2-nitrobenzyl alcohol (0.3
g, 1.96 mmol) were mixed in pyridine (10 mL) at 0 °C. The
ice bath was removed, and the solution was stirred for 15 h.
The mixture was poured into 50 mL of aqueous NaHCO3 (10%)
and extracted with 2 × 10 mL of ether. The aqueous layer
was acidified with dilute HCl to pH 1 and the product
extracted with chloroform. Most of the solvent was evapo-
rated, and the product 17 crystallized on standing at -22 °C:
1
mp 98 °C; yield 46%; H NMR (CDCl3) δ 12.8-11.5 (br, 1 H),
7.92 (d, J ) 8.1 Hz, 2 H), 7.72 (d, J ) 7.7 Hz, 2 H), 7.50 (t, J
) 7.7 Hz, 2 H), 7.30 (t, J ) 7.7 Hz, 2 H), 5.34 (d, J ) 6.7 Hz,
4 H); 13C NMR (D2O/acetone-d6) δ 148.0, 135.4, 134.1 (J CP
)
8.23 Hz), 130.1, 129.9, 125.8, 65.9 (J CP ) 3.47 Hz); 31P NMR
(CD3CN/D2O) δ 1.99 (s). Anal. Calcd for C14H13N2O8P: C,
45.66; H, 3.56; N, 7.61. Found: C, 45.62; H, 3.60; N, 7.64.
P h otolysis. Gen er a l Meth od s. Irradiations for the UV
spectroscopic detection of the nitrosobenzaldehyde photoprod-
Ack n ow led gm en t. This work was supported by
IRG-58-34 from the American Cancer Society and by a
grant (MCB-8920118) from the National Science
Foundation.
(22) This compound was synthesized by a procedure similar to one
reported by: Baldwin, J . E.; McConnaughie, A. W.; Moloney, M. G.;
Pratt, A. J .; Shim, S. B. Tetrahedron 1990, 46, 6879-6884.
J O961861M