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J. E. Coughlin et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1783–1786
reaction, the reaction mixture was concentrated, extracted with hexanes to
mixture was incubated at 37 °C in a water-bath for 1 h and at selected time
points, each aliquot was treated with 200 L methanol and adjusted to pH 6.0 by
the addition of 500 L of NH4OAc (0.1 M, pH 7.0) and 85 L of NaOH (0.1 N). After
standing for 5 min, the incubate was centrifuged, the supernatant concentrated
in a speed vac, diluted with 300 L of NH4OAc (0.1 M, pH 7.0) and analyzed by
reversed-phase HPLC [Waters Instrument equipped with 600E gradient
remove the excess of iodomethyl compounds, lyophilized and purified using
column chromatography. Alternatively, the reaction mixture after hexane
extraction could be directly taken for HPLC purification. Yields of the purified
prodrugs ranged from 44% to 51%. 31P NMR, DMSO-d6, d ppm: 2, 28.1, 28.9; 3,
27.7, 28.6 ppm.
l
l
l
l
a
9. Halogen exchange reactions of chloromethyl esters were carried out with
excess of sodium iodide (2 equiv) in anhydrous acetonitrile at rt overnight in
the dark. After removal of the solvent, the reaction mixture was extracted
between water and DCM, the DCM layer was washed with sodium bisulfite
solution (5%), followed by brine, and dried over anhydrous sodium sulfate. The
organic layer was concentrated to give the iodomethyl esters; also see: Iyer, R.
P.; Yu, D.; Ho, N.-H.; Agrawal, S. Synth. Commun. 1995, 25, 2739.
10. For a review, see: Beaucage, S. L.; Iyer, R. P. Tetrahedron 1993, 49, 1925.
11. (a) Padmanabhan, S.; Coughlin, J. E.; Iyer, R. P. Tetrahedron Lett. 2005, 46, 343;
The LOTUS ReactorÒ is a new chemical reaction chamber designed and built by
us for solid-phase synthesis; see: (b) Iyer, R. P.; Coughlin, J. E.; Padmanabhan, S.
Org. Prep. Proc. Intl. 2005, 37, 205; (c) Iyer, R. P.; Regan, J. B.; Egan, W.; Beaucage,
S. L. J. Am. Chem. Soc. 1990, 112, 1253.
12. Recently, we have also developed efficient solution-phase synthesis, which
provides access to larger quantities of 1 in each batch run (unpublished
results).
13. Hydrolytic rates of Rp,Sp isomers of prodrug analogs 2–3, and the dinucleotide
esters 8–10 were monitored in rabbit serum, according to previously reported
protocols,5 by quantifying the individual Rp, and Sp isomers remaining
unhydrolyzed after 3 h. As can be seen, the hydrolytic rate of Rp, Sp isomers
is clearly R1-substituent dependent. However, the universal applicability of
these observations requires the comparative study of additional analogs.
controller and a 996 photodiode array detector with MILLENNIUM software]. An X-
terra MS C-18 2.5 mm, 2.1 ꢁ 20 mm column and a gradient of 100% A to 100% B
over 30 min of buffer A (0.1M, NH4OAc) and buffer B (80:20, CH3CN/NH4OAc)
with a flow rate of 1 mL/min was employed. The retention times (Rt) were: Rp,Sp
1, 14.3, and 14.6; Rp,Sp 2, 17.8; and Rp,Sp 3, 18.4 min, respectively.
17. The decomposition of 1 in SGF was also monitored by 31P NMR. For this
purpose, initially, the 31P NMR spectrum of 1 in D2O (d 58.6, 58.8 ppm) was
recorded and spectra obtained at different time points following the sequential
addition of pepsin and DCl as per the SGF composition in Ref. 15 A significant
decrease in the peak intensities at d 58.6, 58.8 ppm was noted after 1 h thereby
suggesting decomposition of 1 in SGF.
18. Shafiee, M.; Deferme, S.; Villard, A.-L.; Egron, D.; Gosselin, G.; Imbach, J.-L.;
Lioux, T.; Pompon, A.; Varray, S.; Aubertin, A.-M.; Van Den Mooter, G.; Kinget,
R.; Périgaud, C.; Augustijns, P. J. Pharm. Sci. 2001, 90, 448.
19. Plasma and liver samples were processed and analyzed as before.5
20. de Vrueh, R. L. A.; Smith, P. L.; Lee, C.-P. J. Pharmacol. Exp. Ther. 1998, 286, 1166.
21. The pharmacodynamic studies were conducted at USU (JDM, PI). For the
pharmacodynamic studies,4b homozygous male and female transgenic HBV
mice were used. The mice were originally obtained from Dr. Frank Chisari
(Scripps Research Institute, LaJolla, CA) and were subsequently raised in the
Biosafety Level 3 (BL-3) area of the AAALAC-accredited USU Laboratory Animal
Research Center (LARC). Pre-experiment liver biopsies of mice were obtained
and assayed for liver HBV DNA and subsequently block-randomized to the
treatment groups (8 animals per group). The compounds were reconstituted
with 0.05 M citric acid, pH 2.4 and the final dosing concentrations were such
that 0.1 mL was delivered into a 20 g mouse. The animals were dosed orally
once daily for 14 days. Following the final dosing, the animals were euthanized
and the liver isolated, processed for Southern blot analysis to quantitate liver
HBV DNA. Hybridization of the blots was carried out using a [32P]CTP-labeled,
HBV genomic probe (digested with Hae III) cloned into the pBluescript plasmid
(gift of Dr. Luca Guidotti, The Scripps Institute, LaJolla, CA) overnight at 60 °C in
a solution of 10% PEG-8000, 0.05 M NaPO4, 0.21 mg/ml salmon sperm DNA, and
7% SDS. The radioactive signals were measured using the phosphorimaging
method (Optiquant). An image of the radioactive filter was exposed to a
Cyclone™ Storage Phosphor Screen (Perkin Elmer Multisensitive Medium, Type
MS PPN 7001723). The exposed screen was transferred to the Cyclone™ drum
and read using the 600 dpi setting. The data was stored on a computer, and the
density of the bands was measured. The ratio of the viral DNA bands to the
transgene band was used to determine the concentration of viral DNA per host
DNA. This calculation was based upon the knowledge that there were 1.3
copies of the transgene present per host cell with this line of transgenic mice
(personal communication to JDM from F. Chisari). The transgene was used as
Structure
Compd R
R1
X
B1 B2 Rp:Sp Rp:Sp
t = 0 t = 3 h
2
3
8
9
–OCH3 — Ade Ura 47:52 —*
HO
S
B2
O
–OCH3
O
Ade Ura 49:50 41:58
R1
H
H
— Ade Thy 36:63 1:98
O
P
O
O
X
O
O
O
Ade Thy 47:52 10:89
Thy Thy 60:39 1:98
R
B1
O
O
10
H
OH H
*
Complete hydrolysis occured at both isomers.
14. For excellent reviews on the concept of pseudorotation in the hydrolysis of
cyclic phosphates, see: (a) Westheimer, F. H. Acc. Chem. Res. 1968, 1, 70; (b)
Singleton, R. J., Jr. J. Chem. Educ. 1973, 50, 538.
15. The United States Phamacopeia, 23rd ed. The National Formulary 18: p 2053,
1994 Rockville, MD.
an internal indicator to calculate the pg of HBV DNA per
DNA. The pg HBV DNA/ g host DNA was calculated.
lg of cellular host
16. Stability studies in SGF and SIF: 10
lL of each prodrug (2 mg in 200 lL of DMSO)
l
were diluted with 190 L of either SGF or SIF (prepared according to Ref. 15). The
l