Design and synthesis of novel bis(l-amino acid) ester prodrugs of 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA) with improved anti-HBV activity

Article abstract of DOI:10.1016/j.bmcl.2006.10.021

A series of novel bis(l-amino acid) ester prodrugs of 9-[2-(phosphonomethoxy)ethyl] adenine (PMEA) was synthesized and their anti-HBV activity was evaluated in HepG 2 2.2.15 cells. Compounds 11, 12, 21, 22, 26, and 27 demonstrated more potent anti-HBV act

Full text of DOI:10.1016/j.bmcl.2006.10.021

Bioorganic & Medicinal Chemistry Letters 17 (2007) 465–470
Design and synthesis of novel bis(L-amino acid) ester prodrugs
of 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA)
with improved anti-HBV activity
Xiaozhong Fu,a Saihong Jiang,a Chuan Li,a Jian Xin,b Yushe Yanga,* and Ruyun Jia
aState Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institute for Biological Sciences,
Chinese Academy of Science, Shanghai 201203, China
bShanghai Fudan-Yueda Bio-Tech Co., Ltd, Shanghai 201203, China
Received 13 July 2006; revised 28 September 2006; accepted 10 October 2006
Available online 12 October 2006
Abstract—A series of novel bis(L-amino acid) ester prodrugs of 9-[2-(phosphonomethoxy)ethyl] adenine (PMEA) was synthesized
and their anti-HBV activity was evaluated in HepG 2 2.2.15 cells. Compounds 11, 12, 21, 22, 26, and 27 demonstrated more potent
anti-HBV activity and higher selective index (SI) than adefovir dipivoxil, which was used as a positive control. Compound 11, which
was found to be the most potent one, was five times more potent than adefovir dipivoxil with EC50 value of 0.095 lM and CC50
value of 6636 lM. The SI value (>69,000) of compound 11 was 60 times and 24 times higher than those of adefovir dipivoxil
and lamivudine, respectively. In vitro stability studies showed that compound 11 was relatively more stable than adefovir dipivoxil
with t1/2 of 270 min. These findings suggested that compound 11 could be considered as a promising candidate for further in vivo
studies.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Hepatitis B virus (HBV) can cause both acute and
chronic infection. Of the approximately two billion peo-
ple who have been infected with HBV worldwide, an
estimated 350–400 million are chronically infected, with
0.5–1.2 million deaths annually from the resulting cir-
rhosis, liver failure, and hepatocellular carcinoma.1 Only
a few drugs are currently approved by the FDA for the
treatment of chronic HBV infection. They include lami-
vudine, adefovir dipivoxil, and entecavir. Lamivudine
has a low sustained response rate, and drug resistance
limits its efficacy.2 Adefovir dipivoxil (1), an ester pro-
drug of PMEA, has potent in vitro and in vivo activity
against HBV. In particular, it has shown impressive
ability to suppress replication of HBV resistance to lam-
ivudine, emtricitabine, and famcicloir.2 However, dose-
limiting nephrotoxicity and its potential of releasing tox-
ic formaldehyde and pivalic acid are its primary limita-
tions.3,4 In order to optimize cellular uptake and
antiviral activity, and to reduce cytotoxicity of PMEA,
several prodrugs such as bis(SATE)-PMEA,5 cyclo Sal-
PMEA,6 and Hep-direct prodrug remofovir (2)7 have
been reported. These new prodrugs have improved the
pharmacodynamics and pharmacokinetics of PMEA,
but their obvious weakness of instability and cytotoxic-
ity6,7 makes the development of newer and more effec-
tive prodrug strategies imperative.
An active transporting mechanism has been applied in the
designing of nucleoside L-amino acid ester prodrugs such
as L-valacyclovir (3)8 and L-valganciclovir (4).9 These
prodrugs significantly enhance oral bioavailability and
anti-viral activity of the parent drugs. This property has
been ascribed to their advantages of being able to be effi-
ciently delivered by the human peptide transporter
1(hPepT1).10,11 Unlike nucleoside agents, acyclic nucleo-
side phosphonates have the advantage of skipping the
requisite first phosphorylation step to reach their active
metabolic form. We reasoned that incorporation of
appropriate amino acids into the PMEA would produce
a new class of prodrugs with improved anti-HBV activity,
bioavailability, and reduced cytotoxicity. To our best
knowledge, the L-amino acid ester prodrug strategy has
not yet been reported in the case of acyclic nucleoside
phosphonates. In this article, we report our efforts related
to the synthesis ofa series ofnovel bis(L-aminoacid) esters
Keywords: Adefovir; Anti-HBV nucleotide; Amino acid; Prodrug.
*
Corresponding author.
ysyang@mail.shcnc.ac.cn
Tel.:
+86
21
50806786; e-mail:
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.10.021

466
X. Fu et al. / Bioorg. Med. Chem. Lett. 17 (2007) 465–470
prodrugs of PMEA as anti HBV agents, and evaluations
of their anti-HBV activity in HepG2 2.2.15 cells, resulting
in the discovery of a promising prodrug 11 with more po-
tent anti-HBV activity, lower cytotoxicity, and higher
selective index (SI) than those of adefovir dipivoxil.
DCC and DMAP yielded 2-bromoethyl ester of N-Boc
-L-amino acids,12 which were coupled with PMEA
using N,N0-dicyclohexyl-4-morpholine carboxamidine
(DCMC) as an acid scavenger in anhydrous DMF to
yield compounds 5–8. Removal of the N-Boc-protecting
group in 4N hydrochloride-1,4-dioxane solution affor-
ded compounds 9–12 as hydrochloride salts (Scheme 1).
NH2
N
O
N
N
N
N
N
N
HN
H2N
The procedure for synthesis of compounds 18–22 is
outlined in Scheme 2. After activation by isobutyl
chloroformate (IBCF), N-Boc-L-amino acids reacted
with H2S at ꢀ20 to ꢀ10 ꢁC to give N-Boc-L-amino
monothioacids, which then reacted with 1,2-dibromoe-
thane, using NaH as base, to give 2-bromoethyl ester
of N-Boc-L-amino monothioacids.13 The ester then
reacted with PMEA to afford compounds 13–17.
Deprotection of the N-protecting group via a proce-
dure similar to that in Scheme 1 generated the desired
compounds 18–22.
O
O
P
O
O
O
O
O
O
NH2
O
O
O
1
3
O
N
NH2
N
Cl
N
N
HN
H2N
N
N
N
OH
O
P
O
O
O
O
O
NH2
O
2
4
Condensation of chloromethyl chlorosulfate with
N-Boc-protected L-amino acids in a dichloromethane–
water mixture using n-Bu4NHSO4 as phase-transfer
catalyst14 smoothly afforded chloromethyl ester of
N-Boc-L-amino acid. The resulting ester then reacted
with PMEA to yield compounds 23–25. Removal of
Synthesis of these novel compounds was carried out in a
straightforward manner. Thus condensation of N-Boc-
L-amino acids with 2-bromoethanol in the presence of
NH2
N
N
NH2
X
N
O
P
O
N
O
O
R
n
O
NH2
R
X
n = 1,2
O
X= O,S
R=Alkyl, H
NH2
O
O
a
b
N
N
N
N
R
R
Br
OH
O
O
O
P
NHBoc
R
O
NHBoc
O
O
NHBoc
O
O
R
O
NHBoc
NH2
N
5-8
c
N
N
N
O
5 R= -CH3
O
R
O
P
6 R=-CH(CH3)2
7 R=-CH(CH3)C2H5
8 R=-CH2C6H5
O
O
NH2
O
O
R
O
NH2
9-12
9 R= -CH3
10 R=-CH(CH3)2
11 R=-CH(CH3)C2H5
12 R=-CH2C6H5
Scheme 1. Reagents and conditions: (a) 2-bromoethanol, DCC, DMAP, CH2Cl2, rt; (b) PMEA, DCMC, DMF, rt–80 ꢁC; (c) 4 N HCl-1,4-dioxane
solution, rt.

X. Fu et al. / Bioorg. Med. Chem. Lett. 17 (2007) 465–470
467
O
O
O
a
c
b
R
Br
R
R
N
S
SH
NHBoc
OH
NHBoc
NHBoc
NH2
NH2
N
N
N
O
O
N
O
P
N
N
O
P
N
d
R
R
O
O
O
O
S
S
NHBoc
NH2
O
O
O
O
R
R
S
S
NHBoc
NH2
13-17
18-22
13 R=-H
18 R=-H
19 R= -CH3
14 R= -CH3
15 R=-CH(CH3)2
20 R=-CH(CH3)2
16 R=-CH(CH3)C2H5
17 R=-CH2C6H5
21 R=-CH(CH3)C2H5
22 R=-CH2C6H5
Scheme 2. Reagents and conditions: (a) H2S, IBCF, NMM, THF, 1 N HCl, ꢀ20 to ꢀ10 ꢁC; (b) 1,2-dibromoethane, NaH, THF, ꢀ20 ꢁC to rt; (c)
PMEA, DCMC, DMF, rt to 80 ꢁC; (d) 4N HCl-1,4-dioxane solution, rt.
N-Boc-protecting group as before gave the desired
compounds 26–28 (Scheme 3).
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 were analyzed using Icy-
cler IQ 3.0. EC50, CC50, and SI of these compounds are
reported in Table 1. Adefovir dipivoxil and lamivudine
were used as positive controls. As indicated in Table 1,
all synthesized compounds demonstrated potent anti-
HBV activity with EC50 values of 0.0952–12.5 lM.16
Compounds 11, 12, 21, 22, 26, and 27 exhibited more
potent anti-HBV activity than adefovir dipivoxil with
EC50 values of 0.095, 0.31, 0.21, 0.21, 0.30, and
0.34 lM, respectively. The most active one, compound
11, was almost as potent as lamivudine and five times
more potent than adefovir dipivoxil. The SI value of
The structure of the target compounds was confirmed
by NMR (1H, 13C, 31P), HRMS, and elemental
analysis.
The synthesized compounds were evaluated for their
inhibitory effect on the replication of HBV in HepG2
2.2.15 cell lines according to the reference.15 Viral
DNA recovered from the secreted particles in cultured
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-
NH2
O
O
N
N
a
b
R
R
NHBoc
R
OH
O
Cl
N
O
P
O
N
O
O
O
NHBoc
NHBoc
O
NHBoc
R
O
O
NH2
N
N
N
N
23-25
c
NH2
O
P
23 R=-CH(CH3)2
24 R=-CH(CH3)C2H5
25 R=-CH2C6H5
O
O
O
R
O
O
NH2
O
O
R
26-28
26 R=-CH(CH3)2
27 R=-CH(CH3)C2H5
28 R=-CH2C6H5
Scheme 3. Reagents and conditions: (a) chloromethyl chlorosulfate, n-Bu4NHSO4, NaHCO3, CH2Cl2–H2O, rt; (b) PMEA, DCMC, DMF, rt to
80 ꢁC; (c) 4N HCl-1,4-dioxane solution, rt.

468
X. Fu et al. / Bioorg. Med. Chem. Lett. 17 (2007) 465–470
Table 1. Anti-HBV evaluation of bis(L-amino acid)ester prodrugs of PMEA
NH2
N
N
NH2
N
O
P
N
O
X
O
R
n
O
O
NH2
nX
R
O
b
c
f
t1/2 (min)
Compounda
R
X
n
EC50 (lM)
CC50 (lM)
SId
clogPe
9
10
Methyl
O
O
O
O
S
2
2
2
2
2
2
2
2
2
1
1
1
12.5
1840
2587
6636
2590
259
146
ꢀ1.75
0.100
1.16
ND
120
270
15
Isopropyl
2-Methypropyl
Benzyl
1.31
1974
69523
8354
25
11
12
0.0952
0.311
10.2
1.08
18
19
H
Methyl
ꢀ2.16
ꢀ1.54
0.310
1.37
ND
ND
ND
ND
ND
ND
ND
ND
45
S
1.56
2415
28712
3409
10984
3378
8560
15378
540
1548
38167
16129
52727
11094
25111
23312
1044
2850
20
21
Isopropyl
2-Methylpropyl
Benzyl
S
0.752
0.211
0.208
0.304
0.341
0.659
0.517
0.0807
S
22
26
S
1.29
Isopropyl
2-Methylpropyl
Benzyl
O
O
O
—
—
ꢀ0.460
0.590
0.510
1.39
27
28
Adefovir dipivoxil
Lamivudine
—
—
—
—
230
—
—
a Obtained as hydrochloride salts.
b EC50 (Concentrations of compounds achieving 50% inhibition of cytoplasmic HBV-DNA synthesis).
c CC50 (Concentrations of compounds required for 50% extinction of HepG2 2.2.15 cells).
d SI (selective index CC50/EC50).
e Calculated using Chemdraw ultra 8.0 program.
f In human plasma, and ‘ND’ indicates not determined.
compound 11 was over 60 times and 24 times higher
than those of adefovir dipivoxil and lamivudine, respec-
tively. The SAR research showed that the compounds
with the larger bulk of the amino acid alkyl group,
11, 12, 21, and 22, showed higher anti-HBV activity
and SI than that of the corresponding compounds 9,
10, 18, and 19 with the smaller bulk of the amino acid
alkyl group. In addition, respective comparisons of 9,
10 with 19, 20, and 11, 12 with 21, 22 indicated that
substitution of the amino acid esters with their corre-
sponding thioesters greatly improve the anti-HBV
activity and SI value of the compounds with a smaller
bulk of amino acid alkyl moiety. However, similar
trend was not observed for compounds with a larger
bulk of amino acid alkyl moiety. Further study of com-
pounds 10, 11, 12, 26, 27, and 28 revealed that structure
modification of amino acids moiety had less effect on
the anti-HBV activity and SI of the bis(L-amino acid)
acetal esters of PMEA.
ter with L-amino acid ester is crucial to antiviral activity
and cell toxicity. These results suggested that bis(L-ami-
no acid) ester prodrugs of PMEA contrast with many
prodrugs of PMEA, which gained antiviral potency
and were accompanied by a proportional enhancement
of cytotoxicity.
The usefulness of the prodrugs of PMEA should de-
pend not only on the stability of the prodrug for its
transport across the cell membrane but also upon its
reversion to the parent compound intracellulary, espe-
cially in the virally infected cells. The half-life (t1/2) of
hydrolysis of the esters was therefore determined in
human plasma.17 Data in Table 1 indicated that com-
pounds 10, 11, and 12 were susceptible to the action
of plasma esterases with t1/2 ranging from 15 to
270 min. The results suggested that compound 11
was more stable than adefovir dipivoxil with t1/2 of
270 min, a length over six times as long as that of
adefovir dipivoxil (t1/2 45 min).
All synthesized compounds, except for compound 18,
had much lower cytotoxicities compared with those of
adefovir dipivoxil and lamivudine. Compounds 11, 20,
22, 27, and 28 exhibited preferable cytotoxicity with
CC50 values of 6636, 28712, 10984, 8560, and
15378 lM. L-Amino acid ester prodrugs possessed sig-
nificantly reduced cell toxicity and higher anti-HBV
activity compared with those of adefovir dipivoxil, sug-
gesting that replacement of bis (pivaloyloxy methyl) es-
In summary, we described a novel class of L-amino acid
ester prodrugs of PMEA as an anti-HBV agent with
very potent activity and reduced toxicity. The results
suggested that L-amino acid ester strategy has significant
potential in the acyclic nucleoside phosphonate prodrug
design. Further biological and SAR studies of this new
class of anti-HBV agents are ongoing in our
laboratories.

X. Fu et al. / Bioorg. Med. Chem. Lett. 17 (2007) 465–470
469
Procedure (2): to a 100 mL two-necked flask were added
PMEA (0.10 g, 0.36 mmol), N,N0-dicyclohexyl-4- morpho-
line carboxamidine (0.21 g, 0.72 mmol), (2S, 3S)-2-bromo-
References and notes
1. Lavanchy, D. J. Viral Hepat. 2004, 11, 97.
2. Chen, Z.; Zheng, M. Expert Opin. Ther. Patents 2005, 15,
1027.
ethyl 3-methyl-2-(pivalamido)
pentanoate (0.58 g,
1.80 mmol), and anhydrous DMF (15 mL). The heteroge-
neous mixture was stirred at room temperature for 24 h
then at 80 ꢁC 5 h. The insolubles were filtered off, and the
filtrate was concentrated in vacuo. The residue was
partitioned between 1% citric acid aqueous solution
(5 mL) and acetic ether (5 mL), the organic layer was
separated, and the aqueous layer was extracted with acetic
ether (2· 5 mL). The combined organic extracts were
washed with water (5 mL) then with brine (5 mL), dried
over anhydrous Na2SO4, filtered, and concentrated in
vacuo. The residue was purified by column chromatogra-
phy on silica gel using a mixture of CH2Cl2 and MeOH
(25:1) to obtain compound 7 (0.052 g, 18.35%) as a white
foam solid. 1H NMR (400 MHz, CDCl3): d 8.32 (s, 1H, 8-
H), 7.96 (s, 1H, 2-H), 5.87 (br s, 2H, NH2), 5.37 (d,
J = 7.86 Hz, 1H, NHCO), 5.21 (d, J = 7.86 Hz, 1H,
NHCO), 4.39 (2H, t, J = 5.32 Hz, NCH2), 4.10–4.37 (m,
10H, 2· CH2CH2OP, 2· CH2CH2OP, 2· COCH), 3.93 (t,
J = 5.26 Hz, 2H, OCH2CH2N), 3.81 (d, J = 8.41 Hz, 2H,
OCH2P), 1.84 (m, 2H, 2· CH(Me)), 1.42 (s, 18H,
2· C(Me)3), 1.05–1.26 (m, 4H, 2· CH2CH3), 0.85–0.91
(m, 12H, 4· CH3). MS (EI) m/z (%): 787 (M+, 12), 163
(100). Anal. Calcd for C34H58N7O12: C, 51.84; H, 7.36; N,
12.45. Found: C, 51.79; H, 7.59; N, 12.16.
3. Wang, J.-T.; Choi, J.-R. Drugs Future 2004, 29, 163.
4. Douglsh, H. S.; Kevin, T. B.; Sanjay, K. N. Am. J.
Physiol. Renal. Physiol. 2001, 281, F197.
5. Benzaria, S.; Pelicano, H.; Johnson, R.; Maury, G.;
Imbach, J.-L.; Aubertin, A.-M.; Obert, G.; Gosselin, G.
J. Med. Chem. 1996, 39, 4958.
6. Meris, C.; Gorbig, U.; Muller, C.; Balzarini, J. J. Med.
Chem. 2005, 48, 8079.
7. 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.
8. Friedrichsen, G. M.; Chen, W.; Begtrup, M.; Lee, C. P.;
Smith, P. L.; Borchardt, R. T. Eur. J. Pharm. Sci. 2002,
16, 1.
9. Sugawara, M.; Hung, W.; Fei, Y. J.; Leibach, V. G.;
Ganapathy, M. E. J. Pharm. Sci. 2000, 89, 781.
10. Carsten, U. N.; Birger, B.; Flemming, S. J.; Sven, F.;
Bente, S. Expert Opin. Ther. Patents 2002, 12, 1329.
11. Isabel, R. A.; Hannelore, D. Trends Pharmacol. Sci. 2003,
23, 434.
12. Chaim, G.; Yakir, K. Tetrahedron Lett. 1979, 20, 3811.
13. Ludwig, B.; Suresh, K. S.; Ghosh, B. K. J. Org. Chem.
1965, 30, 949.
14. Naoyuki, H.; Mitsuya, H.; Takashi, T.; Takayuki, K.;
Tomiki, H.; Kenji, T. Synth. Commun. 1994, 24, 767.
15. Mary, A. S.; Cheng, M. L.; George, A. Proc. Natl. Acad.
Sci. U.S.A. 1987, 84, 1005.
16. Experimental details and characterization data.
(a) General. Tetrahydrofuran was dried by distillation
from sodium/benzophenone. N,N-Dimethylformamide
was dried by distillation in vacuo from calcium hydride.
Other commercially available chemicals and solvents were
used without further purification. NMR spectra were
recorded on a Varian Mercury-400 MHz spectrometer.
Low resolution mass spectra were obtained using a
Finnigan MAT 95 mass spectrometer, and high resolution
mass spectra were obtained using a Kratos MS80 mass
spectrometer. Elemental analysis for carbon, hydrogen,
and nitrogen was determined on a Vario EL elemental
analyzer. Flash chromatography was carried out on silica
gel (200–300 mesh), and chromatographic solvent propor-
tions are expressed on a volume:volume basis.
Procedure (3): to a 10 mL two-necked flask were added
compound 7 (0.21 g, 0.27 mmol), 1,4-dioxane (1 mL), and
4 N hydrochloride-1, 4-dioxane solution (1.13 mL,
4.53 mmol). The mixture was stirred at room temperature
for 4 h. After solvent was removed under vacuum, the
resultant residue was dissolved in ethanol (0.5 mL), and the
solution was stirred at 0 ꢁC for 15 min. Ethyl ether
(2.0 mL) was added to the solution and further stirred at
0 ꢁC for 30 min. The resultant product was collected by
filtration, washed quickly with ethyl ether, and dissolved in
water (0.5 mL) dried by lyophilization to get a white foam
of the compound 11 as hydrochloride salts (0.15 g,
20
93.15%). ½aꢁD þ 14:2 (c 0.60, MeOH). 1H NMR
(400 MHz, CD3OD): d 8.38 (s, 1H, 8-H), 8.42 (s,1H, 2-
H), 4.55 (t, J = 5.08 Hz, 2H, NCH2), 4.38–4.53 (m, 4H,
2· CH2CH2OP), 4.21–4.36 (m, 4H, 2· CH2CH2OP), 4.06–
4.11 (m, 2H, 2· COCH), 3.98–4.02 (m, 4H, OCH2P,
OCH2CH2N), 2.00–2.02 (m, 2H, 2· CH(Me)), 1.34 and
1.57 (m, 4H, 2· CH2CH3), 0.96–1.06 (m, 12H, 4· CH3).
13C NMR (100 MHz, CD3OD): d 170.3 (2C), 152.2, 151.0,
146.6, 145.8, 120.2, 72.7, 66.6 (2C), 66.1 (2C), 65.1, 58.8
(2C), 45.5, 38.3 (2C), 27.2 (2C), 15.0 (2C), 12.6 (2C). 31P
(400 MHz, CD3OD): d 24.0. MS (EI) m/z (%): 587 (M+,
11), 86 (100). HRMS (EI): Calcd for C24H42N7O8P:
587.2832. Found: 587.2840 (M+). Anal. Calcd for
C24H42N7O8P.3HCl.2H2O: C, 39.31; H, 6.68; N, 13.37.
Found: C, 39.17; H, 6.91; N, 13.16.
(b) Detail synthesis and spectroscopic data of compound
11.
Procedure (1): DCC (2.27 g, 0.011 mol) was added slowly
to a solution of N-Boc-L-isoleucine (2.31 g, 0.01 mol), 2-
bromoethanol (1.24 g, 0.01 mol) and DMAP (1.23 g,
0.01 mol) in CH2Cl2 (30 mL) at room temperature. The
reaction mixture was stirred at room temperature for 24 h.
The N,N0-dicyclohexyl urea was filtered off, and the filtrate
was evaporated to leave a residue which was dissolved in
acetic ether, cooled at ꢀ10 ꢁC for 6 h, and the resultant
solid was filtered off. The filtrate was concentrated in
vacuo, and the residue was purified by column chroma-
tography on silica gel with eluent (10:1 petroleum ether/
acetic ether) to get (2S,3S)-2-bromoethyl 3-methyl-2-
(pivalamido) pentanoate as a colorless oil (2.48 g, 76.8%).
1H NMR (400 MHz, CDCl3): d 4.99 (d, J = 8.15 Hz, 1H,
NHCO), 4.48–4.37 (m, 2H, OCH2CH2Br), 4.28 (m, 1H,
COCH), 3.52 (t, J = 6.07 Hz, 2H, OCH2CH2Br), 1.87 (q,
J = 4.71 Hz, 1H, CH(Me)), 1.42 (s, 9H, C(Me)3), 1.22–1.27
(m, 2H, CH2CH3), 0.94–0.87 (m, 6H, 2· CH3). MS (EI) m/
z (%): 323 (M+, 15), 57 (100).
(c) Spectroscopic data for other newly synthesized com-
pounds.
20
Compound 9: ½aꢁD þ 1:4 (c 0.350, MeOH). 1H NMR
(400 MHz, CD3OD): d 8.42 (s, 1H, 8-H), 8.38 (s, 1H, 2-H),
4.55 (t, J = 5.08 Hz, 2H, NCH2), 4.43 (t, J = 4.41 Hz, 4H,
2· CH2CH2OP), 4.32 (m, 4H, 2· CH2CH2OP), 4.18 (q,
J = 7.14 Hz, 2H, 2· COCH), 4.01–4.04 (m, 4H, OCH2P,
OCH2CH2N), 1.56 (d, J = 7.28 Hz, 6H, 2· CH3). MS (EI)
m/z (%): 503 (M+, 0.5), 143 (100).
20
Compound 10: ½aꢁD þ 11 (c 0.286, MeOH). 1H NMR
(400 MHz, CD3OD): d 8.41 (s, 1H, 8-H), 8.36 (s, 1H, 2-H),
4.54 (t, J = 4.76 Hz, 2H, NCH2), 4.40–4.47 (m, 4H,
2· CH2CH2OP), 4.31 (m, 4H, 2· CH2CH2OP), 4.19 (m,
2H, 2· COCH), 3.98–4.05 (m, 4H, OCH2P, OCH2CH2N),

470
X. Fu et al. / Bioorg. Med. Chem. Lett. 17 (2007) 465–470
2.30–2.33 (m, 2H, 2· CH(Me)2), 1.13 (d, J = 6.96 Hz, 6H,
12.5 (2C). MS (ESI) m/z (%): 620.2 (MH+, 100). HRMS
(ESI): Calcd for C24H42N7O6PS2Na: 642.2273. Found:
642.2319 (MNa+).
2· CH3), 1.06 (d, J = 7.06 Hz, 6H, 2· CH3). 31P (400 MHz,
CD3OD): d 27.1. MS (EI) m/z (%): 559 (M+, 0.7), 72 (100).
Anal. Calcd for C22H38N7O8P.3HCl.2H2O: C, 37.47; H,
6.38; N, 13.91. Found: C, 37.29; H, 6.33; N, 13.79.
1
Compound 22: H NMR (400 MHz, CD3OD): d 8.38 (s,
1H, 8-H), 8.35 (s, 1H, 2-H), 7.28–7.38 (m, 10H, ArH), 4.56
(m, 2H, NCH2), 4.05–4.18 (m, 6H, 2· CH2CH2OP,
2· COCH), 3.96 (t, J = 4.59 Hz, 2H, OCH2CH2N), 3.91
(d, J = 8.25 Hz, 2H, OCH2P), 3.24 (m, 4H, 2· SCH2CH2),
3.13–3.19 (m, 4H, 2· CH2Ph). 13C NMR (100 MHz,
CD3OD): d 197.9 (2C), 152.9, 151.6, 146.4, 142.4, 135.6
(2C), 131.4 (4C), 131.0 (4C), 129.8 (2C), 120.2, 72.7, 66.3
(2C), 65.1, 57.2 (2C), 45.4, 39.1 (2C), 31.0 (2C). MS (ESI)
m/z (%): 688.2 (MH+, 100). HRMS (ESI): Calcd for
C30H39N7O6PS2: 688.2141. Found: 688.2161 (MH+).
20
Compound 12: ½aꢁD þ 7:2 (c 0.304, MeOH). 1H NMR
(400 MHz, CD3OD): d 8.37 (s, 1H, 8-H), 8.35 (s, 1H, 2-H),
7.25–7.36 (m, 10H, ArH), 4.51 (t, J = 5.09 Hz, 2H, NCH2),
4.27–4.41 (m, 8H, 2· CH2CH2OP, 2· CH2CH2OP), 4.22
(m, 2H, 2· COCH), 3.98–4.01 (m, 4H, OCH2P,
OCH2CH2N), 3.16–3.28 (m, 4H, 2· CH2Ph). MS (EI) m/
z (%): 655 (M+, 1.2),178 (100).
Compound 18: 1H NMR (400 MHz, DMSO-d6): d 8.49 (s,
1H, 8-H), 8.42 (s, 1H, 2-H), 4.54 (t, J = 5.12 Hz, 2H,
NCH2), 4.31 (m, 4H, 2· CH2CH2OP), 3.97 (d, J = 7.82 Hz,
2H, OCH2P), 3.92 (m, 2H, OCH2CH2N), 3.85 (s, 4H,
2· COCH2), 3.26 (m, 4H, 2 · SCH2CH2). 13C NMR
(100 MHz, CD3OD): d 169.1 (2C), 152.3, 150.9, 146.6,
146.0, 120.1, 72.7, 66.5 (2C), 66.2, 56.7 (2C), 41.6, 31.3 (2C).
MS (ESI) m/z (%): 508.3 (MH+, 100). HRMS (ESI): Calcd
for C16H27N7O6PS2: 508.1124. Found: 508.1139 (MH+).
20
Compound 26: ½aꢁD þ 7:4 (c 0.253, MeOH). 1H NMR
(400 MHz, CD3OD): d 8.42 (s, 1H, 8-H), 8.37 (s, 1H, 2- H),
5.65–5.91 (m, 4H, 2· OCH2OP), 4.57 (t, J = 4.89 Hz, 2H,
NCH2), 4.11 (m, 2H, 2· COCH), 4.08 (d, J = 8.22 Hz, 2H,
OCH2P), 4.02 (t, J = 4.89 Hz, 2H, OCH2CH2N), 2.14–2.21
(m, 2H, 2· CH(Me)2), 1.10 (d, J = 5.48 Hz, 6H, 2· CH3),
1.07 (d, J = 5.28 Hz, 6H, 2· CH3). MS (ESI) m/z (%): 532.1
(MH+, 100). Anal. Calcd for C20H34N7O8PÆ3HClÆ1H2O: C,
36.45; H, 5.92; N, 14.88. Found: C, 36.17; H, 6.18; N, 15.16.
20
Compound 19: ½aꢁD þ 2:1 (c 0.270, MeOH). 1H NMR
(400 MHz, CD3OD): d 8.43 (s, 1H, 8-H), 8.38 (s, 1H, 2-H),
4.55 (t, J = 5.13 Hz, 2H, NCH2), 4.34 (q, J = 6.97 Hz, 2H,
2· COCH), 4.16–4.20 (m, 4H, 2· CH2CH2OP), 4.02–3.98
(m, 4H, OCH2P, OCH2CH2N), 3.29 (m, 4H,
2· SCH2CH2), 1.61 (d, J = 6.96 Hz, 6H, 2· CH3). 13C
NMR (100 MHz, CD3OD): d 198.4 (2C), 152.7, 151.1,
146.5, 146.3, 120.1, 72.7, 66.5 (2C), 64.9, 56.9 (2C), 45.5,
30.9 (2C), 18.1 (2C). MS (ESI) m/z (%): 536.1 (MH+, 100).
HRMS (ESI): Calcd for C18H30N7O6PS2Na: 558.1334.
Found: 558.1359 (MNa+).
1
Compound 27: H NMR (400 MHz, CD3OD): d 8.41 (s,
1H, 8-H), 8.36 (s, 1H, 2-H), 5.73–5.89 (m, 4H,
2· OCH2OP), 4.56 (t, J = 5.13 Hz, 2H, NCH2), 4.18 (m,
2H, 2· COCH), 4.08 (d, J = 8.07 Hz, 2H, OCH2P), 4.01 (t,
J = 5.14 Hz, 2H, OCH2CH2N), 2.05 (m, 2H, 2· CH(Me)),
1.32 and 1.61 (m, 4H, 2· CH2CH3), 0.97–1.06 (m, 12H,
4· CH3). 31P (400 MHz, CD3OD): d 23.6. 13C NMR
(100 MHz, CD3OD): d 169.6 (2C), 152.1, 151.0, 146.6,
145.7, 120.0, 84.6 (2C), 72.8, 66.9, 58.7 (2C), 45.4, 38.2 (2C),
27.1 (2C), 15.4 (2C), 12.5 (2C). MS (ESI) m/z (%): 560.2
(MH+, 100). HRMS (ESI): Calcd for C22H39N7O8P:
560.2519. Found: 560.256 (MH+).
20
Compound 20: ½aꢁD þ 16:1 (c 0.267, MeOH). 1H NMR
(400 MHz, CD3OD): d 8.43 (s, 1H, 8-H), 8.39 (s, 1H, 2-H),
4.58 (t, J = 4.69 Hz, 2H, NCH2), 4.14–4.28 (m, 6H,
2· CH2CH2OP, 2· COCH), 3.98–4.02 (m, 4H, OCH2P,
OCH2CH2N), 3.31 (t, J = 6.26 Hz, 4H, 2· SCH2CH2),
2.26–2.41 (m, 2H, 2· CH(Me)2), 1.12 (d, J = 6.85 Hz, 6H,
2· CH3), 1.04 (d, J = 7.04 Hz, 6H, 2· CH3). MS (ESI) m/z
(%): 592.1 (MH+, 100).
20
Compound 28: ½aꢁD þ 12:5 (c 0.277, MeOH). 1H NMR
(400 MHz, CD3OD): d 8.35 (s, 1H, 8-H), 8.34 (s, 1H, 2-H),
7.37–7.25 (m, 10H, ArH), 5.85–5.70 (m, 4H, 2· OCH2OP),
4.53–4.46 (m, 4H, NCH2, 2· COCH), 4.09 (d, J = 7.70 Hz,
2H, OCH2P), 4.01 (t, J = 5.13 Hz, 2H, OCH2CH2N), 3.21–
3.15 (m, 4H, 2· CH2Ph). 13C NMR (100 MHz, CD3OD): d
169.7 (2C), 152.4, 151.3, 146.5, 146.0, 135.8 (2C), 131.1
(4C), 130.8 (4C) 129.5 (2C), 119.6, 84.5 (2C), 72.6, 67.3,
58.2 (2C), 45.5, 37.8 (2C). MS (ESI) m/z (%): 628.2 (MH+,
100). HRMS (ESI): Calcd for C28H35N7O8P: 628.2206.
Found: 628.2217 (MH+).
20
Compound 21:½aꢁD þ 28 (c 0.208, MeOH). 1H NMR
(400 MHz, CD3OD): d 8.41 (s, 1H, 8-H), 8.36 (s, 1H, 2-H),
4.56 (t, J = 5.12 Hz, 2H, NCH2), 4.18–4.25 (m, 6H,
2· CH2CH2OP, 2· COCH), 3.91–4.01 (m, 4H, OCH2P,
OCH2CH2N), 3.24 (m, 4H, 2· SCH2CH2), 2.07 (m, 2H,
2· CH(Me)), 1.24 and 1.61 (m, 4H, 2· CH2CH3), 0.97–1.08
(m, 12H, 4· CH3). 13C NMR (100 MHz, CD3OD): d 197.2
(2C), 152.2, 151.4, 146.4, 142.4, 119.7, 72.7, 66.6 (2C), 65.4,
57.7 (2C), 45.5, 38.8 (2C), 31.1 (2C), 26.4 (2C), 15.6 (2C),
17. Agarwal, S. K.; Gogu, S. R.; Rangan, S. R. S.; Agrawal,
K. C. J. Med. Chem. 1990, 33, 1505.