Design, synthesis and evaluation of novel oxazaphosphorine prodrugs of 9-(2-phosphonomethoxyethyl)adenine (PMEA, adefovir) as potent HBV inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.10.072

A series of novel oxazaphosphorine prodrugs of 9-(2-phosphonomethoxyethyl)adenine (PMEA, adefovir) were synthesized and their anti-hepatitis B virus (HBV) activity was evaluated in HepG2 2.2.15 cells, with adefovir dipivoxil as a reference drug. In the ce

Full text of DOI:10.1016/j.bmcl.2009.10.072

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6918–6921
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and evaluation of novel oxazaphosphorine prodrugs
of 9-(2-phosphonomethoxyethyl)adenine (PMEA, adefovir)
as potent HBV inhibitors
a
Peng Lu a, Jiangxia Liu b, Yuya Wang a, Xiaoyan Chen a, Yushe Yang a, , Ruyun Ji
*
a State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institute for Biological Sciences, Chinese Academy of Science, Shanghai 201203, China
b Key Laboratory of Medical Molecular Viology, Institute of Medical Microbiology, Shanghai Medical College, Fudan University, Shanghai 200032, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel oxazaphosphorine prodrugs of 9-(2-phosphonomethoxyethyl)adenine (PMEA, adefovir)
were synthesized and their anti-hepatitis B virus (HBV) activity was evaluated in HepG2 2.2.15 cells, with
adefovir dipivoxil as a reference drug. In the cell assays, compounds 7b and 7d exhibited anti-HBV activ-
Received 30 August 2009
Revised 14 October 2009
Accepted 16 October 2009
Available online 21 October 2009
ity comparable to that of adefovir dipivoxil, while compound 7c, with an IC50 value of 0.12 lM, was found
to be three times more potent than the reference compound. In vitro stability studies showed that (SP,S)-
7c, the diastereomer of compound 7c, was stable in human blood plasma but underwent rapid metabo-
lism to release the parent drug PMEA in liver microsomes. The possible metabolic pathway of (SP,S)-7c in
human liver microsomes was described. These findings suggest that compound (SP,S)-7c is a promising
anti-HBV drug candidate for further development.
Keywords:
Oxazaphosphorine
Prodrug
Hepatitis B virus
Ó 2009 Elsevier Ltd. All rights reserved.
Hepatitis B virus (HBV) infection is a major global health prob-
lem. Over 350 million people worldwide are infected chronically
with HBV and are therefore at risk of various liver diseases, which
results in 0.5–1.2 million deaths each year (the tenth leading cause
of PMEA prodrugs including acyloxyalkyl esters, S-acyl-2-thioethyl
(SATE) esters8 and phosphoramidates9 have been developed.
Although promising, these prodrugs are cleaved by esterases,
which results in their rapid breakdown in the blood, and a lack
of sustained therapeutic effects. In this Letter, we report our efforts
to develop a series of oxazaphosphorine prodrugs of PMEA 7a–i
(Fig. 1) with more potent anti-HBV activity, lower cytotoxicity
and better plasma stability.
Synthesis of oxazaphosphorine prodrugs of PMEA 7a–i is out-
lined in Scheme 1. N-(3-Hydroxypropyl)-amino acid esters 4a–i
were prepared from various commercially available amino acids
in 25–34% yields in five steps, according to a previously described
method, with minor modifications.10 N-Protected PMEA dichloride
3 was obtained by treating PMEA with oxalyl chloride in the pres-
ence of N,N-diethylformamide in refluxing CH2Cl2, followed by
activation with two equivalents of pyridine at 0 °C in CH2Cl2.11
Cyclization of 4a–i with phosphonyl dichlorides 3 at ꢀ78 °C, fol-
lowed by removal of the imine protecting group with acetic acid
yielded cyclized oxazaphosphorines 6a–i as oils. Treatment of
6a–i with one equivalent of fumaric acid in refluxing acetonitrile
for 2 h provided the final products 7a–i as crystalline salt, with
yields of 15–33% for all four steps.12
of death worldwide).1 Although interferon
a and several nucleo-
side and nucleotide analogues currently are available for the treat-
ment of HBV, they are associated with a low cure rate, viral
resistance, cytotoxicity and other limitations.2 New drugs with
better therapeutic profiles are still urgently needed.
Among current anti-HBV agents,3 nucleotide analog 9-(2-phos-
phonomethoxyethyl)adenine (PMEA) (1, Fig. 1) has shown impres-
sive activity against a broad spectrum of DNA viruses including
HBV, and more importantly, it is associated with a low rate of viral
resistance. However, the negative charge of the phosphonate moi-
ety significantly impairs its cellular uptake and leads to poor oral
bioavailability.4 One potential strategy for overcoming these limi-
tations is to use prodrugs.5 One of the best examples is the prodrug
adefovir dipivoxil (2, Fig. 1), which has been approved by the US
FDA for HBV therapy. Adefovir dipivoxil can be converted rapidly
into the parent drug PMEA in vivo and suppress replication of
HBV that is resistant to other anti-HBV drugs such as lamivudine,
emtricitabine and famciclovir.6 However, the dose-limiting neph-
rotoxicity of adefovir and its potential to release the carcinogen
formaldehyde and toxic pivalic acid have limited its clinical use.7
To circumvent these therapeutic limitations, several new classes
To investigate stereochemistry effects of the phosphine groups
on anti-HBV activity, four diastereomerically pure analogs were
prepared. As shown in Scheme 2, chromatographic resolution of
5g afforded diastereomers (SP,S)-5g and (RP,S)-5g. (SP,S)-7g and
(RP,S)-7g were prepared in two steps from (SP,S)-5g and (RP,S)-5g,
respectively. Using 5i as the starting material, (RP,R)-7i and
* Corresponding author. Tel./fax: +86 21 50806786.
E-mail address: ysyang@mail.shcnc.ac.cn (Y. Yang).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.10.072

P. Lu et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6918–6921
6919
Figure 1. Structures of PMEA (1), adefovir dipivoxil (2) and compounds 7a–i.
Scheme 1. Reagents and conditions: (a) (i) Et2NCHO, (COCl)2, CH2Cl2, reflux, 3 h, (ii) Py, 0 °C, 20 min; (b) TEA, CH2Cl2, ꢀ78 °C; (c) AcOH, reflux, 2 h; (d) fumaric acid, MeCN,
reflux, 1 h.
Scheme 2. Reagents: (a) chromatographic resolution; (b) AcOH, reflux, 2 h; (c) fumaric acid, MeCN, reflux, 1 h.
(SP,R)-7i were obtained using a similar method to that described
for compound 7g. The spectroscopic data of 1H and 31P NMR were
used to assign the configuration of the four diastereomers of 7g
and 7i.13 The results are summarized in Table 1.
The target compounds were evaluated for their inhibitory ef-
fects on the replication of HBV in HepG2 2.2.15 cells, as described
previously.14 Viral DNA recovered from the secreted particles in
culture medium was analyzed by real-time PCR using Icycler
(Bio-Rad). Amplification primers were HBVFP: 50-TGT CCT GGT
TAT CGC TGG-30 and HBVRP: 50-CAA ACG GGC AAC ATA CCT T-30.
The TaqMan probe was FAM-50-TGT GTC TGC GGC GTT TTA TCA
T-30-TAMRA. The data was analyzed using Icycler IQ 3.0. IC50
,

6920
P. Lu et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6918–6921
Table 1
porated into oxazaphosphorine prodrugs of PMEA 7a–i. In L-amino
acid series, all the compounds except 7d showed less cytotoxicity
Analytical data of diastereomers of compounds 7g and 7i
than adefovir dipivoxil did (Table 2), which suggests that introduc-
Compound
MS
NMR d (ppm)
1H (C-20, CH3)b
tion of an
cytotoxicity, probably because of production of relatively less toxic
metabolites. Incorporation of the unnatural amino acid -valine led
to production of prodrug 7i, which was more toxic than adefovir
dipivoxil. -valine prodrug 7c and -leucine prodrug 7d exhibited
potent anti-HBV activity, with IC50 values of 0.12 and 0.32 M,
L-amino acid ester moiety at the N-3 position reduces
1H (C-10, CH3)b
31Pc
(SP,S)-7g
(RP,S)-7g
(RP,R)-7i
(SP,R)-7i
463.1824a
463.1838a
463.1834a
463.1815a
0.94
0.82
0.94
0.82
0.86
0.65
0.86
0.65
21.77
22.02
21.76
21.95
D
L
L
l
HRMS (FAB), m/z calcd for C18H29N6O5P [M+Na]+ 463.1835.
a
1H chemical shift was recorded at 300 MHz with a Varian Gemini spectrometer
b
respectively. The most potent compound 7c was nearly three
using tetramethylsilane as an internal standard.
times more active than the reference compound adefovir dipivoxil.
C
31P chemical shift was recorded at 121 MHz using 85% H3PO4 in a coaxial insert
D-valine prodrug 7i showed no inhibition of HBV replication.
as an external standard.
From these results, it appeared that the contribution of amino acid
moieties to anti-HBV activity has the following order of magnitude:
L-valine >
L-leucine >
L-alanine > glycine >
L-isoleucine > L-phenylala-
Table 2
nine > >
D
-valine. The poor activity of
D
-valine prodrug 7i suggests
Anti-HBV activity of prodrugs 7a–i
that the configuration of amino acid is an important determinant
of anti-HBV activity.
R1
R2
IC50
(lM)
CC50 (lM)
SIc
a
b
Compound
Since the L-valine methyl ester 7c emerged as the most active
7a
H (rac)
Me
Me
Me
Me
Me
Me
Me
Me
Me
Et
0.88
0.51
0.91
0.12
0.32
2.21
2.62
3.00
50
602
650
590
577
222
648
726
560
468
592
439
400
555
683
1270
649
4800
694
293
277
187
9
(SP,S)-7b
(RP,S)-7b
7c
7d
7e
compound, the effect of ester groups on anti-HBV activity was
examined further. As shown in Table 2, although ethyl ester 7g
and isopropyl ester 7h showed a similar CC50 value to that of the
methyl ester prodrug 7c, their potency as indicated by their IC50
values was diminished 25- and 48-fold, respectively. The de-
creased inhibitory activity was possibly caused by the increased
steric hindrance of the ester groups.
To explore the influence of stereochemistry on anti-HBV activ-
ity, six diastereomerically pure compounds were obtained by
successive applications of column chromatography and crystalliza-
i-Pr (S)
i-Bu (S)
s-Bu (S)
Bn (S)
i-Pr (S)
i-Pr (S)
i-Pr (S)
i-Pr (R)
i-Pr (R)
7f
(SP,S)-7g
(RP,S)-7g
7h
(RP,R)-7i
(SP,R)-7i
Et
i-Pr
Et
5.76
50
50
103
9
8
Et
Adefovir dipivoxil
0.33
1680
a
tion. As shown in Table 2,
inhibition of HBV replication. For the
logs, (SP,S)-7b (IC50 = 0.51 M) and (RP,S)-7b (IC50 = 0.91
was a modest preference for the S-phosphor diastereomer. The
same trend was observed for the -valine ethyl ester 7g in that
(SP,S)-7g displayed more potent anti-HBV activity than (RP,S)-7g.
D
-valine (SP,S)-7i and (RP,S)-7i showed no
-alanine methyl ester ana-
M), there
IC50 (Concentrations of compounds achieving 50% inhibition of cytoplasmic
L
HBV-DNA synthesis).
b
CC50 (Concentrations of compounds required for 50% extinction of HepG2 2.2.15
l
l
cells).
c
SI (selectivity index) = CC50/IC50
.
L
CC50 and SI of these compounds are listed in Table 2. Adefovir dip-
ivoxil was used as a positive control.
Based on the data in Table 2,
further profiling.
L-valine prodrug 7c was chosen for
In our previous Letter, we reported a series of L-amino acid ester
It has been reported that adefovir dipivoxil has poor in vitro sta-
bility in plasma, which causes rapid decomposition and decreased
therapeutic index.16 Thus, the in vitro stability of 7c in human
blood and liver microsomes was examined further. Both diastereo-
mers of 7c were stable in plasma (t1/2 >6 h), whereas adefovir
prodrugs of PMEA with enhanced antiviral activity.15 It is thought
that amino acid esters can be delivered efficiently by human pep-
tide transporter 1. To take advantage of the active transporting
mechanism of amino acids, various amino acid esters were incor-
Figure 2. Possible metabolic pathway of (SP,S)-7c.

P. Lu et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6918–6921
6921
(2 equiv, 0.32 mL, 4.0 mmol). The cold solution was then added slowly to a
ꢀ78 °C solution of N-(3-hydroxypropyl)-amino acid esters (4a–i, 1 equiv,
2 mmol) and triethylamine (6.3 equiv, 1.8 mL, 12.6 mmol) in CH2Cl2 (20 mL) at
dipivoxil displayed poor stability with a half-life of 45 min in hu-
man plasma and microsomes. Although (RP,S)-7c and (SP,S)-7c have
similar stability in plasma, the hydrolysis rate of isomer (SP,S)-7c
(t1/2 = 3.6 h) in human microsomes was faster than that of (RP,S)-
7c (t1/2 >6 h). Moreover, diastereomer (SP,S)-7c was more stable
than adefovir dipivoxil in human blood and liver microsomes.
The possible metabolic pathway for prodrug (SP,S)-7c was
investigated further in human microsomes (Fig. 2). Prodrug
(SP,S)-7c was incubated with human liver microsomes for 6 h and
the metabolites were identified with LC–MS. Oxidation of the car-
bon adjacent to the amide led to the intermediate 8 (LC–MS peak at
12.5 min with an observed mass of 443). Subsequent hydrolysis of
8 afforded the second intermediate 9, which underwent a facile
b-elimination to produce the third intermediate 11 (LC–MS peak
at 17.1 min with an observed mass of 387) and acrolein 10, a
known glutathione scavenger.17 Furthermore, 11 was hydrolyzed
to the parent compound PMEA, which underwent subsequent
phosphorylation to form triphosphate as an HBV polymerase
inhibitor.18 The parent compound PMEA appeared in LC–MS at
4.1 min, which was confirmed by comparison with the authentic
sample. Compound 11 was found to be the major metabolite in hu-
man microsomes.
In summary, we described the synthesis and structure–activity
relationships of a novel class of oxazaphosphorine prodrugs of
PMEA, as highly potent anti-HBV agents with excellent stability
in human plasma. The possible metabolic pathway in human
microsomes for the most potent prodrug 7c was described. The re-
sults suggest that oxazaphosphorine prodrugs of PMEA have signif-
icant potential for treatment of HBV infection. A further study of
the prodrug (SP,S)-7c is underway in our laboratory and will be re-
ported in due course.
a
rate that maintained the internal reaction temperature at ꢀ78 °C. The
reaction mixture was warmed to 0 °C for 2 h, washed with water (3 ꢁ 20 mL),
and the organic layer was dried over MgSO4 and concentrated under reduced
pressure. The resultant residue was purified by chromatography (CH2Cl2/
MeOH, 20:1) to give N-protected oxazaphosphorine prodrugs of PMEA as white
foams.The above prepared foam was dissolved in ethanol (30 mL). Acetic acid
(2.2 equiv, 0.25 mL, 4.4 mmol) was added, and the solution was heated at
reflux for 2 h. The reaction mixture was cooled to room temperature and
concentrated under reduced pressure. The resultant residue was purified by
chromatography (CH2Cl2/MeOH, 20:1) to give 6a–i as colorless oils.
The oils obtained were redissolved in acetonitrile (10 mL) and treated with
appropriate fumaric acid (1 equiv). After being refluxed for 2 h, the solution
was allowed to cool to room temperature for 2 h. The solid was filtered, rinsed
with ether (5 mL) and dried to give 7a–i fumaric acid salts as crystalline solids.
The first-eluting diastereoisomer (SP,S)-7g: mp 128–129 °C. ½aꢂD = ꢀ3.2° (c
0.340, MeOH). 1H NMR (CD3OD, 300 MHz) d 8.21 (s, 1H, 20-H), 8.16 (s, 1H, 80-H),
6.75 (s, 2H, CH@CH), 4.46 (m, 2H, NCH2), 4.23 (m, 2H, OCH2), 3.84–4.08 (m, 6H,
OCH2P + OCH2 + OCH2CH3), 3.53 (t, J = 10.2 Hz, 1H, NCHCO), 3.06 (m, 2H,
NCH2), 2.17 (m, 1H, CHH), 1.96 (m, 1H, CHH), 1.67 (m, 1H, CH(CH3)2), 1.18 (t,
J = 7.2 Hz, 3H, OCH2CH3), 0.94 (d, J = 6.6 Hz, 3H, CH3), 0.86 (d, J = 6.6 Hz, 3H,
CH3). 13C NMR (CD3OD, 75 MHz) d 172.2 (2C), 168.9, 157.5, 153.8, 151.2, 143.7,
135.8 (2C), 120.4, 72.7, 71.1, 69.5, 68.0, 65.0, 62.4, 45.1, 43.0, 28.0, 27.5, 20.1,
19.7, 15.0. 31P NMR (CD3OD, 120 MHz) d 21.77. MS (ESI) m/z 441.3 [M+H]+,
463.3 [M+Na]+. HRMS calcd for C18H29N6O5P [M+Na]+ 463.1824, found
463.1835.
The second-eluting diastereoisomer (RP,S)-7g: mp 231–232 °C. ½aꢂD = ꢀ18.0° (c
0.300, MeOH). 1H NMR (CD3OD, 300 MHz) d 8.21 (s, 1H, 20-H), 8.19 (s, 1H, 80-H),
6.75 (s, 2H, CH@CH), 4.50 (m, 2H, NCH2), 4.08–4.29 (m, 4H, OCH2 + OCH2CH3),
3.77–3.94 (m, 4H, OCH2P + CH2O), 3.53 (t, J = 9.9 Hz, 1H, NCHCO), 3.40 (m, 2H,
NCHH), 3.05 (m, 2H, NCHH), 1.96 (m, 2H, CH2), 1.62–1.89 (m, 1H, CH(CH3)2),
1.26 (t, J = 7.2 Hz, 3H, OCH2CH3), 0.82 (d, J = 6.6 Hz, 3H, CH3), 0.65 (d, J = 6.6 Hz,
3H, CH3). 13C NMR (CD3OD, 75 MHz) d 173.8 (2C), 168.8, 157.4, 153.5, 151.3,
143.8, 135.8 (2C), 120.4, 72.7, 71.4, 69.4, 67.8, 65.4, 62.3, 45.2, 42.9, 29.3, 27.9,
20.2, 20.1, 14.9. 31P NMR (CD3OD, 120 MHz) d 22.02. MS (ESI) m/z 441.3
[M+H]+, 463.3 [M+Na]+. HRMS calcd for C18H29N6O5P [M+Na]+ 463.1838, found
463.1835.
The first-eluting diastereoisomer (RP,R)-7i: mp 176–177 °C. ½aꢂD = +2.9° (c
0.340, MeOH). 1H NMR (CD3OD, 300 MHz) d 8.21 (s, 1H, 20-H), 8.16 (s, 1H, 80-H),
6.75 (s, 2H, CH@CH), 4.46 (m, 2H, NCH2), 4.23 (m, 2H, OCH2), 3.84–4.08 (m, 6H,
OCH2P + OCH2 + OCH2CH3), 3.53 (t, J = 10.2 Hz, 1H, NCHCO), 3.00–3.13 (m, 2H,
NCH2), 2.17 (m, 1H, CHH), 2.01 (m, 1H, CHH), 1.67 (m, 1H, CH(CH3)2), 1.18 (t,
J = 7.2 Hz, 3H, OCH2CH3), 0.94 (d, J = 6.6 Hz, 3H, CH3), 0.86 (d, J = 6.6 Hz, 3H,
CH3). 13C NMR (CD3OD, 75 MHz) d 172.2 (2C), 168.9, 157.5, 153.8, 151.2, 143.7,
135.8 (2C), 120.4, 72.7, 71.1, 69.5, 68.0, 65.0, 62.4, 45.1, 43.0, 28.0, 27.5, 20.1,
19.7, 15.0. 31P NMR (CD3OD, 120 MHz) d 21.76. MS (ESI) m/z 441.3 [M+H]+,
463.3 [M+Na]+. HRMS calcd for C18H29N6O5P [M+Na]+ 463.1834, found
463.1835.
References and notes
1. Malik, A.; Lee, W. Ann. Intern. Med. 2000, 132, 723.
2. (a) Wong, D. K. H.; Cheung, A. M.; O’Rourke, K.; Naylor, C. D.; Detsky, A. S.;
Heathcote, J. Ann. Intern. Med. 1993, 119, 312; (b) Lok, A. S.; Hussain, M.;
Cursano, C.; Margotti, M.; Gramenzi, A.; Grazi, G. L.; Jovine, E.; Benardi, M.;
Andreone, P. Hepatology 2000, 32, 1145; (c) Kim, J. W.; Park, S. H.; Louie, S. G.
Ann. Pharmacother. 2006, 40, 472.
3. (a) De Clercq, E.; Sakuma, T.; Baba, M.; Pauwels, R.; Balzarini, J.; Rosenberg, I.;
Holy, A. Antiviral Res. 1987, 8, 261; (b) Yokota, T.; Mochizuki, S.; Konno, K.;
Mori, S.; Shigheta, S.; De Clercq, E. Antimicrob. Agents Chemother. 1991, 35, 394.
4. Ulrika, E.; John, M. H.; Jae-Seung, K.; Stefanie, M.; Paul, K.; Katherine, Z. B.; Julie,
M. B.; John, C. D.; Boris, A. K.; Charles, E. M. Bioorg. Med. Chem. Lett. 2007, 17,
583.
5. (a) Krise, J. P.; Stella, V. J. Adv. Drug Delivery Rev. 1996, 19, 287; (b) Schultz, C.
Bioorg. Med. Chem. 2003, 11, 885.
6. (a) Chen, Z.; Zheng, M. Exp. Opin. Ther. Patents 2005, 15, 1027; (b) Douglas, H. S.;
Kevin, T. B.; Sanjay, K. N. Am. J. Physiol. Renal Physiol. 2001, 281, F197.
7. Speit, G.; Schutz, P.; Merk, O. Mutagenesis 2000, 15, 85.
The second-eluting diastereoisomer (SP,R)-7i: obtained as a white solid. mp
136–137 °C. ½aꢂD
= +31.1° (c 0.325, MeOH). 1H NMR (CD3OD, 300 MHz) d 8.21
(s, 1H, 20-H), 8.19 (s, 1H, 80-H), 6.75 (s, 2H, CH@CH), 4.50 (m, 2H, NCH2), 4.08–
4.29 (m, 4H, OCH2 + OCH2CH3), 3.77–3.94 (m, 4H, OCH2P + CH2O), 3.53 (t,
J = 9.9 Hz, 1H, NCHCO), 3.41 (m, 2H, NCHH), 2.95 (m, 2H, NCHH), 1.96 (m, 2H,
CH2), 1.62–1.89 (m, 1H, CH(CH3)2), 1.26 (t, J = 7.2 Hz, 3H, OCH2CH3), 0.82 (d,
J = 6.6 Hz, 3H, CH3), 0.65 (d, J = 6.6 Hz, 3H, CH3). 13C NMR (CD3OD, 75 MHz) d
173.8 (2C), 168.8, 157.4, 153.5, 151.3, 143.8, 135.8 (2C), 120.4, 72.7, 71.4, 69.4,
67.8, 65.4, 62.3, 45.2, 42.9, 29.3, 27.9, 20.2, 20.1, 14.9. 31P NMR (CD3OD,
120 MHz) d 21.99. MS (ESI) m/z 441.3 [M+H]+, 463.3 [M+Na]+. HRMS calcd for
C18H29N6O5P [M+Na]+ 463.1815, found 463.1835.
13. (a) Pikul, S.; Dunham, K. L. M.; Almstead, N. G.; De, B.; Natchus, M. G.;
Anastasio, M. V.; McPhail, S. J.; Snider, C. E.; Taiwo, Y. O.; Chem, L.; Dunaway, C.
M.; Gu, F.; Mieling, G. E. J. Med. Chem. 1999, 42, 87; (b) Sawa, M.; Kiyoi, T.;
Kurokawa, K.; Kumihara, H.; Yamamoto, M.; Miyasaka, T.; Ito, Y.; Hirayama, R.;
Inoue, T.; Kirii, Y.; Nishiwaki, E.; Ohmoto, H.; Maeda, Y.; Ishibushi, E.; Inoue, Y.;
Yoshino, K.; Kondo, H. J. Med. Chem. 2002, 45, 919; (c) Mosbo, J.; Verkade, J. G.
A. J. Org. Chem. 1977, 42, 1549; (d) Cooper, D. B.; Harrison, J. M.; Inch, T. D.;
Lewis, G. J. J. Chem. Soc., Perkin Trans. 1 1974, 1049.
14. Mary, A. S.; Cheng, M. L.; George, A. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 1005.
15. Fu, X. Z.; Jiang, S. H.; Li, C.; Xin, J.; Yang, Y. S.; Ji, R. Y. Bioorg. Med. Chem. Lett.
2007, 17, 465.
16. (a) Farquhar, D.; Srivastva, D. N.; Kattesch, N. J.; Saunders, P. P. J. Pharm. Sci.
1983, 72, 324; (b) Farquhar, D.; Khan, S.; Srivastva, D. N.; Saunder, P. P. J. Med.
Chem. 1994, 37, 3902; (c) Dang, Q.; Brown, B. S.; van Poelje, P. D.; Colby, T. C.;
Erion, M. D. Bioorg. Med. Chem. Lett. 1999, 9, 1505.
8. Lefebvre, I.; Perigaud, C.; Pompon, A.; Aubertin, A. M.; Girarder, J. L.; Kirn, A.;
Gosselin, G.; Imbach, J. L. J. Med. Chem. 1995, 38, 3941.
9. McGuigan, C.; Pathirana, R. N.; Balzarini, J.; De Clercq, E. J. Med. Chem. 1993, 36,
1048.
10. Sorensen, M. D.; Blæhr, L. K. A.; Christensen, M. K.; Hoyer, T.; Latini, S.; Hjarnaa,
P. V.; Björkling, F. Bioorg. Med. Chem. 2003, 11, 5461.
11. (a) Erion, M. D.; Reddy, K. R.; Boyer, S. H.; Matelich, M. C.; Gomez-Galeno, J.;
Lemus, R. H.; Ugarkar, B. G.; Colby, T. J.; Schanzer, J.; Van Poelje, P. D. J. Am.
Chem. Soc. 2004, 126, 5154; (b) Erion, M. D.; Van Poelje, P. D.; Mackenna, D. A.;
Colby, T. J.; Montag, A. C.; Fujitaki, J. M.; Linemeyer, D. L.; Bullough, D. A. J.
Pharmacol. Exp. Ther. 2005, 46, 4321; (c) Hecker, S. J.; Reddy, R.; Van Poelje, P.
D.; Sun, Z.; Varkhedkar, W.; Huang, V.; Reddy, M. V.; Fujitaki, J. M.; Olsen, D. B.;
Koeplinger, K. A.; Boyer, S. H.; MacCoss, D. L.; Linemeyer, M.; Erion, M. D. J. Med.
Chem. 2007, 50, 3891.
12. Experimental details and characterization data. Standard procedure: preparation
of oxazaphosphorine prodrugs of PMEA 7a–i: Oxalyl chloride (3.5 equiv,
0.60 mL, 7.0 mmol) was added slowly to a slurry of PMEA (1.0 equiv, 2.0 mmol)
and N,N-diethylformamide (1.1 equiv, 0.24 mL, 2.2 mmol) in CH2Cl2 (20 mL),
and the resulting mixture was refluxed for 3 h. The reaction was cooled to
room temperature and concentrated in vacuo. The resultant yellow foam was
dissolved in CH2Cl2 (20 mL), cooled to 0 °C, and treated slowly with pyridine
17. Banijamali, A. R.; Xu, Y.; Dematteo, V.; Strunk, R. J.; Sumner, S. J. J. Agric. Food.
Chem. 2000, 48, 4693.
18. (a) Balzarini, J.; Hao, Z.; Herdewjin, P.; Johns, D.; De Clercq, E. Proc. Natl. Acad.
Sci. U.S.A. 1991, 88, 1499; (b) Merta, A.; Votruba, I. J. Biochem. Pharmacol. 1992,
44, 2067; (c) Robbins, B.; Greenhaw, J.; Connelly, M.; Fridland, A. Antimicrob.
Agents Chemother. 1995, 39, 2304.