Synthesis and pharmacokinetic and pharmacodynamic evaluation of the forodesine HCl analog BCX-3040

Article abstract of DOI:10.1080/15257770500267246

□ Forodesine HCl is a potent inhibitor of the enzyme purine nucleoside phosphorylase (PNP) and is currently in clinical trials for the treatment of leukemia and lymphoma. Animal models indicated that forodesine HCl would have low oral bioavailability in h

Full text of DOI:10.1080/15257770500267246

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Synthesis and Pharmacokinetic and Pharmacodynamic
Evaluation of the Forodesine HCl Analog BCX-3040
Hollis S. Kezar III a , J. Michael Kilpatrick a , Deborah Phillips a , Debbie Kellogg a , Jianwen
Zhang a & Philip E. Morris Jr. a
a BioCryst Pharmaceuticals, Inc. , Birmingham, Alabama, USA
Published online: 16 Aug 2006.
To cite this article: Hollis S. Kezar III , J. Michael Kilpatrick , Deborah Phillips , Debbie Kellogg , Jianwen Zhang & Philip E.
Morris Jr. (2005) Synthesis and Pharmacokinetic and Pharmacodynamic Evaluation of the Forodesine HCl Analog BCX-3040,
Nucleosides, Nucleotides and Nucleic Acids, 24:10-12, 1817-1830, DOI: 10.1080/15257770500267246
To link to this article: http://dx.doi.org/10.1080/15257770500267246
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Nucleosides, Nucleotides, and Nucleic Acids, 24:1817–1830, 2005
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Copyright ꢀ Taylor & Francis Group, LLC
ISSN: 1525-7770 print/1532-2335 online
DOI: 10.1080/15257770500267246
SYNTHESIS AND PHARMACOKINETIC AND PHARMACODYNAMIC
EVALUATION OF THE FORODESINE HCl ANALOG BCX-3040
Hollis S. Kezar III, J. Michael Kilpatrick, Deborah Phillips, Debbie Kellogg,
ᮀ
Jianwen Zhang, and Philip E. Morris, Jr.
BioCryst Pharmaceuticals, Inc.,
Birmingham, Alabama, USA
ᮀ
Forodesine HCl is a potent inhibitor of the enzyme purine nucleoside phosphorylase (PNP)
and is currently in clinical trials for the treatment of leukemia and lymphoma. Animal models
indicated that forodesine HCl would have low oral bioavailability in humans and it was initially
developed as an intravenous formulation. We were interested in identifying analogs of forodesine
HCl with improved oral bioavailability. The 2 -deoxy analog (BCX-3040) was synthesized and
its pharmacokinetic and pharmacodynamic properties compared with forodesine HCl.
Keywords Forodesine HCl; Purine nucleoside phosphorylase; PNP; T-cell
INTRODUCTION
Forodesine HCl (BCX-1777) is a novel inhibitor of the enzyme purine
nucleoside phosphorylase (PNP) and is in human clinical trials for the
treatment of leukemia and lymphomas.[1–5] The enzyme purine nucleoside
phosphorylase (PNP, EC 2.4.2.1) catalyzes the reversible cleavage of purine
nucleosides to the corresponding purine base and sugar phosphate in the
purine salvage pathway as shown below.[6]
Dedicated to the memory of John A. Montgomery.
Received 7 February 2005; accepted 6 May 2005.
Address correspondence to Philip E. Morris, Jr., BioCryst Pharmaceuticals, Inc., 2190 Parkway
Lake Drive, Birmingham, AL 35244. E-mail: pmorris@biocryst.com
1817

1818
H. S. Kezar III et al.
In the absence of PNP, nucleoside substrates such as 2ꢁ-deoxyguano-
sine (dGuo) accumulate. dGuo accumulation has been observed in chil-
dren with inherited PNP deficiency and as a consequence, these children
exhibit severe T-cell immunodeficiency but retain normal or exagger-
ated B-cell function.[7] T-cell cytotoxicity is due to phosphorylation of
dGuo (via 2ꢁ-deoxycytidine kinase, dCK, (EC 2.7.1.74)) to 2ꢁ-deoxyguano-
sine triphosphate (dGTP). dGTP allosterically inhibits the enzyme ribonu-
cleotide diphosphate reductase (EC 1.17.4.1), preventing DNA synthesis
and hence T-cell proliferation.[8] The relatively unique sensitivity of T-cells
is attributed to their relatively high level of dCK compared to other cells.
This observation has led to the development of PNP inhibitors for the treat-
ment of T-cell cancers and T-cell autoimmune indications. The biochemi-
cal basis for the use of PNP inhibitors as well as the various classes of in-
hibitors developed has been reviewed.[9,10] The use of forodesine HCl as
an immunosuppresent has also been demonstrated.[11] More recently, clin-
ical trial experience in psoriasis with the PNP inhibitor BCX-34 has been
described.[12]
When bound to the PNP enzyme, forodesine mimics the charged
ribosyl oxocarbenium ion complex formed during the transition state of
the enzyme-catalyzed C-N bond cleavage of nucleosides. Forodesine is a
slow-onset, tight-binding inhibitor with a Ki* of 23 pM and is one of the
most potent inhibitors known for the enzyme.[13] Initial pharmacokinetic
experiments conducted in mice indicated an oral bioavailability of 63%.
However, in rats the oral bioavailability dropped to 13% and in primates
the measured oral bioavailability was 11%.[14] Based on these results,
forodesine HCl was initially developed as an intravenous formulation. As
part of our development program, we were interested in identifying analogs
of forodesine HCl with improved oral pharmacokinetic characteristics.
That dGuo binds tightly to PNP suggests that the 2ꢁ-OH group con-
tributes little to binding and is not required for activity. Based on this obser-
vation, we proposed that the 2ꢁ-deoxy analog (1) of forodesine HCl would
retain the potency of forodesine HCl and would have improved oral bioavail-
ability. In this manuscript the synthesis of 1 is presented with a pharmacoki-
netic and pharmacodynamic comparison to forodesine HCl.

Evaluation of the Forodesine HCl Analog BCX-3040
1819
RESULTS AND DISCUSSION
Chemistry
BCX-3040 (1) was synthesized according to Scheme 1. We have previ-
ously described the synthesis of 4 via addition of the lithiated 9-deazapurine
derivative 2 to the carbohydrate derived cyclic imine 3.[15] With 4 in hand,
we envisioned the title compound 1 could be obtained through a sequence
of selective deprotection, protection and followed by deoxygenation of the
unprotected 2ꢁ-OH group. Previous unpublished work from our laboratory
had shown that under acidic conditions the relative stabilities of the acid
labile protecting groups of 4 was PhCH2OCH2 OCH3 Boc isopropy-
∼
lidene TBDMS. Partial deprotection of 4 was accomplished with 1N HCl
=
in methanol, which selectively cleaved only the more labile TBDMS and iso-
propylidene groups affording the triol 5 in excellent yield. The 3ꢁ and 5ꢁ-OH
groups were protected as the 1,1,3,3-tetraisopropyldisiloxyl ether, which af-
forded 6. The 2ꢁ-OH group was converted to the thiocarbonate 7 in excellent
yield. Deoxygenation was accomplished by heating 7 with 1,1ꢁ-azobis(cyclo-
hexane-1-carbonitrile) in toluene at reflux to give 8. While it is possible
to directly convert 8 to the title compound 1 under acidic conditions we
chose a two-step procedure in which the PhCH2OCH2 (BOM) protecting
group is cleaved first with catalytic hydrogenation followed by acid hydrol-
ysis of the remaining protecting groups. The advantage of this route is it
obviates the need for the strongly acidic conditions required to cleave the
BOM group, which often results in formation of tarry material, which can
be difficult to remove from the final product. Thus 8 was hydrogenated
with 20% Pd(OH)2 in ethanol containing a small amount of NH4OH. The
presence of NH4OH is required since catalytic removal of BOM groups un-
der neutral conditions is sometimes complicated by formation of the inter-
mediate NCH2OH, which easily converted in situ in the presence of base
to the desired NH compound 9. Acid hydrolysis of 9 afforded 1, which
was isolated as the hydrochloride in an overall yield of 40% from 4 over
seven steps.
Biology: A Comparison of thePharmacokinetic and
Pharmacodynamic Properties of BCX-3040 and Forodesine HCl
After a single 5 mg/kg IV dose of BCX-3040 there is a rapid decrease
in plasma BCX-3040 (Figure 1) with a terminal half life ( t½) of 0.97 h
(Table 1). The BCX-3040 clearance, 5.8 mL/min (348 mL/h) (Table 1),
is greater than the rat glomerular filtration rate (1.3 mL/min).[16] The
dGuo response follows a similar pattern (Figure 2) with a slightly delayed
Cmax (Table 1) and a terminal half life of 1.2 h. The normal plasma con-
centration of dGuo is below the limit of detection for both methods used.

1820
H. S. Kezar III et al.
SCHEME 1 Reagents. i. nBuLi/anisole/ether/−70 C; ii. Boc-anhydride/CH2Cl2; iii. 1N HCl/MeOH/
30 C; iv. 1,3-dichloro-1,1-3,3-tetraisopropyldisoloxane/pyridine; v. phenyl chlorothionoformate/MeCN;
vi. 1,1 -azobis(cyclohexane-1-carbonitrile)/toluene reflux; vii. 20% Pd (OH)2, H2, ethanol NH4OH
(trace); viii. HCl/heat.

Evaluation of the Forodesine HCl Analog BCX-3040
1821
FIGURE 1 Concentration time curves of BCX-3040 after 5 mg/kg IV dosing, and 5 and 10 mg/kg
oral dosing of BCX-3040. Shown are the mean standard error bars.
TABLE 1 Pharmacokinetics of BCX-3040 and dGuo in Rats after Single Doses of
5 and 10 mg/kg BCX-3040
Pharmacokinetic parameters BCX-3040
Parameter
Units
Route
Dose
Cmax
Tmax
AUCall
λt½
CLobs
Vz
IV
5
Oral
5
0.0029
0.80
0.0318
ND
—
ND
Oral
10
mg/kg
µg/mL
h
3.0
0.083
3.7
0.97
348.5
537.2
0.31
0.00
0.028
0.086
33.55
32.66
0.018
1.6
0.093
0.078
3.2
0.50
0.0169
0.75
0.114
µg·h/mL
h
ND
—
ND
mL/h
mL
F
n
%
—
3
2.5
0.87
6.8
1.52
4
4
Pharmacokinetic parameters of plasma dGuo
Parameter
Units
Route
Dose
Cmax
Tmax
AUCall
λt½
IV
5
Oral
5
0.00189
1.15
0.006
Oral
10
mg/kg
µg/mL
h
0.40
0.83
1.2
0.045
0.167
0.64
0.005
6.0
0.02
0.041
4.0
0.23
1.3
0.0033
0.00
0.030
0.39
µg·h/mL
h
1.2
0.008
ND
n
3
4
4
ND, Not determined; there were not 3 points available for analysis only 2 for λt½ had
3 points available for analysis, but only one of these had a correlation coefficient greater
than 0.9. Therefore, an accurate value for this parameter could not be determined from
these data.

1822
H. S. Kezar III et al.
FIGURE 2 Concentration time curves of plasma dGuo after 5 mg/kg IV dosing, and 5 and 10 mg/kg
oral dosing of BCX-3040. Shown are the mean standard error bars.
TABLE 2 Pharmacokinetics of Forodesine and dGuo in Rats after Single Doses of
1, 5, and 10 mg/kg Forodesine HCl
Pharmacokinetic parameters forodesine HCl
Parameter
Units
Route
Dose
Cmax
Tmax
AUCall
λt½
Clobs
Vz
IV
1
Oral
5
Oral
10
0.157
1.00
0.27
2.58
—
ND
1.03
mg/kg
µg/mL
h
2.0
0.083
2.6
1.1
101.5
151.4
0.20
0.00
0.54
0.11
17.89
22.53
0.24
3.0
1.4
7.0
0.078
0.20
2.0
1.5
6.8
1.29
0.26
3.25
µg·h/mL
h
mL/h
mL
%
—
ND
1.82
F
N
—
5
10.6
5.6
—
7
7
Pharmacokinetic parameters of plasma dGuo
Parameter
Units
Route
Dose
Cmax
Tmax
AUCall
λt½
IV
1
Oral
5
Oral
10
0.0393
0.94
0.48
0.59
mg/kg
µg/mL
h
0.75
1.6
5.0
3.7
0.197
0.60
1.26
0.71
0.33
8.6
2.9
4.9
0.050
0.35
4.7
3.0
3.8
2.82
0.33
2.00
µg·h/mL
h
N
5
7
7

Evaluation of the Forodesine HCl Analog BCX-3040
1823
FIGURE 3 Concentration time curves of Forodesine after 1 mg/kg IV dosing, and 5 and 10 mg/kg
oral dosing of Forodesine. Shown are the mean standard error bars.
IV dosing of rats with 1 mg/kg forodesine HCl produced a slightly lower
Cmax than did dosing with 5 mg/kg BCX-3040 (Tables 1 and 2, Figures 1
and 3). Both Cmaxs were observed 5 min after the bolus doses and were the
first samples collected. The relatively low BCX-3040 Cmax is probably due to
the more rapid clearance of BCX-3040.
Oral dosing produced mean oral BCX-3040 bioavailabilities of 2.5 and
6.8% for the 5 and 10 mg/kg doses, respectively. The terminal half-life for
BCX-3040 and dGuo after the 5 mg/kg dose could not be determined due
to low plasma concentration of both analytes. For 10 mg/kg oral the mean
terminal half life was 1.3 h. Compared to the dGuo response produced by
forodesine, the dGuo response produced by BCX-3040 was low (Tables 1
and 2, Figures 2 and 4). Using the more sensitive LC/MS/MS method, the
FIGURE 4 Concentration time curves of plasma dGuo after 1 mg/kg IV dosing, and 5 and 10 mg/kg
oral dosing of Forodesine. Shown are the mean
standard error bars.

1824
H. S. Kezar III et al.
dGuo response was barely quantitatable after the 5 mg/kg oral dose of BCX-
3040 and was 8 times less than the corresponding forodesine-dGuo response
after dosing orally with 10 mg/kg BCX-3040.
Both BCX-3040 and forodesine are potent inhibitors of PNP in erythro-
cyte extracts with IC50s of 3.1 0.50 nM and 1.2 0.21 nM12 (mean SE),
respectively. These IC50s are higher than would be found using purified en-
zyme but are sufficient for comparison.
BCX-3040 bioavailability and BCX-3040-dependent dGuo response after
oral administration of BCX-3040 are significantly lower compared to forode-
sine HCl (Table 2). Oral 5 mg/kg forodesine HCl resulted in a 12.6-fold
higher dGuo response than did the 10 mg/kg BCX-3040 oral treatment.
Additionally, plasma terminal terminal half life of forodesine after oral ad-
ministration of 10 mg/kg was 5 times longer than the corresponding oral
dose of BCX-3040. The dGuo response after a single IV dose of 1 mg/kg
forodesine HCl was not quite twofold greater than the 5 mg/kg IV BCX-
3040 dGuo response. This corresponds to their respective plasma Cmaxs and
clearances for the IV dosed groups. The combination of rapid elimination
and poor bioavailability make BCX-3040 a poor in vivo inhibitor of PNP
as related to forodesine HCl. Another factor that could play a role would
be a slow extended absorption of forodesine leading to a longer apparent
half-life.[14] The Cmax and AUC for the oral doses of both BCX-3040 and
forodesine did not increase in proportion to the dose. It is hypothesized
that this may be due to a saturable transporter.[14]
BIOLOGY EXPERIMENTAL
Animal Housing
The rats (male, Sprague Dawley, Charles River) fed and watered ad
libitum. They were housed in a humidity-, light-, and temperature-controlled
environment.
Pharmacokinetic Study Design
BCX-3040 was dosed at 5 mg/kg IV, and 5 and 10 mg/kg orally in
groups of 3, 4, and 4 rats, respectively. Forodesine HCl was dosed IV in
5 rats/group as a bolus at 1 mg/kg IV and given orally in 7 rats/group
at 5 and 10 mg/kg. Previous studies indicated that forodesine dosed IV at
1 mg/mL produced the maximum plasma dGuo response, and preliminary
experiments indicated that 1 mg/mL BCX-3040 was inadequate. Addition-
ally, dosing 5 mg/kg IV and oral with BCX-3040 was used to get a more
accurate indication of bioavailability. For the IV studies blood samples were
collected pre, 5 min, 15 min, 30 min, 1 h, 4 h, 8 h, 12 h, and 24 h of the IV
dose; and for the single dose oral studies blood was collected pre, 30 min,

Evaluation of the Forodesine HCl Analog BCX-3040
1825
1 h, 4 h, 8 h, 12 h, and 24 h following the oral doses. The animals were
weighed immediately before dosing to determine the dose volume.
Blood Collection and Plasma Preparation
For all the studies blood was collected in BCX-34 (to achieve a final
concentration of 50 uM) spiked tubes. This acted as a preservative for dGuo.
Plasma samples were collected after centrifugation of the blood stored at
around −80◦C.
BIOANALYTICAL ANALYSIS
Plasma BCX-3040 and dGuo Analysis
For plasma BCX-3040 and dGuo analysis the determinations were made
within the same run. The plasma concentration of BCX-3040 and dGuo was
determined by LC-MS-MS after ultrafiltration of the plasma using Amicon
Centrifree filters. The filtrates were then analyzed using C-3 (Zorbax SB-C3
2.1 X 40 mm, 5 m) reverse phase HPLC and selected reaction monitoring
(SRM, multiple reaction monitoring, MRM) method of mass spectrometric
detection (Q1/Q3 Masses: 251.30/215.10 amu for BCX-3040 and 268/152
for dGuo). The concentrations of BCX-3040 and dGuo were determined
using standard curves of each prepared in rat plasma. Quality control
samples were distributed throughout the run. A PE Sciex API2000 MS/MS
with a Hewlett-Packard 1100 HPLC were used for the analysis.
Plasma Forodesine and dGuo Analyses
For forodesine and dGuo analysis the determinations were made in sep-
arate methods due to the different chromatographic properties of the two.
Forodesine was determined by LC/MS analysis after solid-phase extraction.
A Zorbax SB C-3, 3.0X150 mm column was used for the chromatography.
An internal standard method was used with quadratic regression analysis,
using inosine-U-15N4 as the internal standard.
Mass Spectrometry, positive electrospray in single ion monitoring mode
was used for BCX-1777 set at 208 m/z with the fragmentor set at 130 volts.
A Hewlett-Packard 1100-MSD was used for the analysis.
dGuo concentrations were determined by HPLC, Hewlett-Packard 1100,
high-performance liquid chromatography system equipped with a binary
pump, solvent degassing module, autosampler, and a UV detector. Plasma
samples were ultrafiltered using Centrifree YM-30 filters as per the man-
ufacturers instructions. The analytes were eluted from a Hewlett-Packard
Purospher RP C-18 12 × 150 mm column with 5% DMSO in 10 mM ammo-

1826
H. S. Kezar III et al.
nium formate, pH 5.0. The flow rate was 0.5 mL/min and the run time was
20 min.
For both assays quality control samples were included throughout the
batch, and standard curves were run in pooled rat plasma that was spiked
with the appropriate analyte prior to extraction or filtration.
PNP Analysis
Inhibition enzymatic activity of PNP in erythrocyte extracts was deter-
mined by monitoring the PNP dependent hydrolysis of inosine spectopho-
tometrically. A Cary 3 Spectrophotometer, Vavian Model 1001206 equipped
with Cary WinUV software was used for these studies. The IC50 was deter-
mined by determining the concentration of compound, which reduced the
rate of inosine hydrolysis by 50% compared to the buffer control. Specifi-
cally, the PNP is prepared from rat erythrocytes after lysis of the erythrocytes
with 0.2% Triton X-100 and adjusted to the required specific activity. Ten L
of inhibitor and water are added to 40 L of PNP containing ∼1 mUnit of
PNP. This is then added to 1450 L of 50 mM phosphate buffer containing
0.5 mM inosine and excess, 80 mU/mL, xanthine oxidase. One unit of PNP
causes the phosphorolysis of 1 mol of inosine to hypoxanthine and ribose
1-phosphate per minute at pH 7.2 at 25◦C. In the presence of excess xan-
thine oxidase, the hypoxanthine is hydrolyzed to uric acid. Generation of
uric acid is monitored at 293 nm. An inhibition curve was generated using
0.625, 1.25, 5, 10, and 50 nM. The concentration of inhibitor that resulted
in 50% inhibition of the rate of generation uric acid is reported as the IC50.
Pharmacokinetic Analysis
Data were reported as mean
S.E. Pharmacokinetic analysis was per-
formed using WINNONLIN Standard 3.1, EXCEL, and Sigma Plot. Non-
compartmental analysis was used to calculate the following parameters:
C
max: maximum observed plasma concentration of BCX-1777 or 2ꢁ-deoxy-
guanosine obtained after the each dose.
t
max: time of maximum observed plasma concentration of BCX-1777 or 2ꢁ-
deoxyguanosine obtained after the each dose.
AUCall: area under the curve for a single dose over a 24-h period.
Lambda z t ½: ( t½) terminal half-life of the terminal elimination phase of
drug disposition.
Clearance (Clobs): millimeters of drug cleared of drug per unit time.
Volume of distribution (Vz): base on the terminal elimination phase; Vz =
dose/ 2 · AUCinf.
Oral bioavailability (F): percent drug absorbed after oral administration
base on the IV and oral AUCall.

Evaluation of the Forodesine HCl Analog BCX-3040
1827
CHEMISTRY EXPERIMENTAL
General
Melting points were determined on a Meltemp II melting point appara-
1
tus and are uncorrected. The H-NMR spectra were recorded on a Bruker
Avance 300 at 300.13 MHz. Chemical shifts (ppm) are referenced to inter-
nal tetramethylsilane and spectra were recorded at ambient temperature. IR
spectra were obtained on a Bio-Rad FTS-7 FT-IR. Mass spectra were recorded
on a Micromass ZMD in the positive electrospray mode with a scan range
of 0–1000 m/z and cone voltage setting of 20 V. A solution of the sam-
∼
ple ( 100 g/mL) in methanol (100%) was introduced into the source via
=
a Waters 2690 autosampler. Flash chromatographic separations were per-
formed on Whatman silica gel, 60 Å or using a Biotage Flash 40i or 75i with
prepacked silica gel (60 Å) cartridges. Thin-layer chromatography (TLC)
was performed using aluminum backed silica gel 60 plates from E. Merck.
Inosine was purchased from Sigma Chemical Company, St. Louis, Missouri;
dGuo was obtained from Reliable Biohemicals, St. Louis, Missouri; and BCX-
1777 (forodesine HCl) and BCX-34 were synthesized at BioCryst. Inosine-
U-15Na was obtained from Cambridge Isotope Laboratories, Sorbax C3 and
Purospher C18 columns from Agilent, and phenyl boronic acid solid-phase
cartridges from Varian. Centrifree YM-30 1 mL Micropartition Devices were
obtained from Millipore.
(1S)-N-tert-Butoxycarbonyl-1 -C-{4-methoxypyrrolo[3,2-d]pyrimidin-9-N-
benzyloxo-methyl)}-1 ,4 -dideoxy-1 ,4 -imino-D-ribitol (5). A sample of the
silyl ether 4 (42.5 g, 64.9 mmol) was dissolved in methanol (1.5 L) under
an Argon atmosphere. To this 1N HCl (∼650 mL) was added. After 1
h an additional 150 mL of 1N HCl was added and solution placed in a
water bath at ∼30◦C. After 1.5 h the solution was cooled in an ice/water
bath and made slightly basic (∼pH 8) with concentrated aqueous NH4OH
(∼60 mL). After concentrating the resulting syrup was partitioned between
chloroform and a small amount water. The organic layer was dried (Na2SO4)
and concentrated to give a crude foam. The crude product was purified
by chromatography (Flash 75, 800 g SiO2, EtOAc) and relevant fractions
combined to give 31.2 g (62.3 mmol, 96%) of 5 as a white foam. An analytical
sample was obtained from a center fraction giving 5 as a white foam. IR
(KBr) 2935, 1695, 1614, 1539, and 1365 cm−1; MS (m/z, ES+) 501.1 (100%);
1H NMR (300 MHz, CDCl3) 8.12 (d, J = 17.5 Hz, 1H), 7.47–7.25 (m,
6H), 5.71 (s, 1H), 5.67 (dd, J = 10.5, 59.6 Hz, 1H), 4.78–4.48 (m, 4H),
4.31–3.96 (m, 3H), 4.07 (s, 3H), 3.68 (d, J = 11.5 Hz, 1H), 3.19 (br s, 1H,
D2O exchangeable), 1.31 and 1.05 (pair s, 9H total). Analysis calculated
for C25H32N4O7: C, 59.99; H, 6.44; N, 11.19. Found: C, 59.91; H, 6.50;
N, 10.89.

1828
H. S. Kezar III et al.
(1S)-N-tert-Butoxycarbonyl-1 -C-{4-methoxypyrrolo[3,2-d]pyrimidin-9-N-
benzyloxo-methyl)}-1 ,4 -dideoxy-1 ,4 -imino-3 ,5 -O-(1,1,3,3-tetraisopropyl-
disiloxa-1,3-diyl)-D-ribitol (6). A sample of the triol 5 (28.5 g, 57.0 mmol)
was dissolved in anhydrous pyridine (250 mL) under an Argon atmosphere.
To this 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (20.0 mL, 62.6 mmol,
1.1 eq.) was added. After 15 h the solution was concentrated azeotroping
with toluene (2 × 200 mL). The resulting residue was partitioned between
chloroform and water. After separating layers the aqueous layer was ex-
tracted with chloroform (2×). The organic layer was dried (Na2SO4) and
concentrated. The crude product was purified by chromatography (Flash
75, 800 g SiO2, gradient 10 → 15 → 25 → 50% EtOAc-hexanes) and rele-
vant fractions combined to give 34.35 g (46.2 mmol, 81%) of 6 as a white
foam. An analytical sample was obtained from a center fraction giving 6 as
a white foam. IR (KBr) 2946, 2868, 1699, 1612, 1538, and 1367 cm−1; MS
(m/z, ES+) 743.2 (100%); 1H NMR (300 MHz, CDCl3) 8.47 (s, 1H), 7.45
(br s, 1H), 7.35–7.23 (m, 5H), 5.76 (br d, J = 9.9 Hz, 1H), 5.58 (d, J = 10.6
Hz, 1H), 5.22 (br s, 2H), 4.50–4.41 (m, 4H), 4.19–4.16 (m, 1H), 4.10 (s,
3H), 3.83 (br s, 1H), 3.25 (br s, 1H, D2O exchangeable), 1.41 (br s, 59H),
1.11–0.96 (m, 28H). Analysis calculated for C37H58N4O8Si2: C, 59.81; H,
7.87; N, 7.54. Found: C, 59.89; H, 7.77; N, 7.52.
(1S)-N-tert-Butoxycarbonyl-1 -C-{4-methoxypyrrolo[3,2-d]pyrimidin-9-N-
(benzyloxo-methyl)}-1 ,4 -dideoxy-1 ,4 -imino-2 -O-[phenoxy(thiocarbonyl)]-
3 ,5 -O-(1,1,3,3-tetra-isopropyldisiloxa-1,3-diyl)-D-ribitol (7). A sample of
the alcohol 6 (10.0 g, 13.4 mmol) and DMAP (4.60 g, 37.7 mmol, 2.8 eq.) was
dissolved in anhydrous acetonitrile (200 mL) under an Argon atmosphere.
To this mixture was added phenyl chlorothionoformate (2.0 mL, 14.8 mmol,
1.1 eq.) in one portion. After 12 h the solution was concentrated and the
resulting residue partitioned between dichloromethane and water. The or-
ganic layer was washed with water (2×), dried (Na2SO4), and concentrated.
The crude product was purified by chromatography (Flash 75, 200 g SiO2,
20% EtOAc-hexanes) and relevant fractions combined to give 10.63 g (12.1
mmol, 90%) of 7 as a white foam. An analytical sample was obtained from
a center fraction giving 7 as a white foam. IR (KBr) 2946, 2868, 1701, 1611,
1539, and 1368 cm−1; MS (m/z, ES+) 879.2 (100%); H NMR (300 MHz,
1
DMSO-d6) 8.38 (s, 1H), 7.77 (s, 1H), 7.51–7.46 (m, 2H), 7.36–7.13 (m,
8H), 6.37 (br s, 1H), 5.79–5.72 (m, 2H), 5.49 (br s, 1H), 5.28 (s, 1H), 4.48
(s, 2H), 4.18–4.13 (m, 2H), 4.05 (s, 3H), 3.63–3.57 (m, 1H), 1.38 (br s, 9H),
1.05–0.88 (m, 28H). Analysis calculated for C44H62N4O9S1Si2: C, 60.11; H,
7.11; N, 6.37. Found: C, 60.42; H, 7.21; N, 6.34.
(1S)-N-tert-Butoxycarbonyl-1 -C-{4-methoxypyrrolo[3,2-d]pyrimidin-9-N-
(benzyloxo-methyl)}-1 ,4 -dideoxy-1 ,4 -imino-2 -deoxy-3 ,5 -O-(1,1,3,3-tetra-
isopropyldisiloxa-1,3-diyl)-D-ribitol (8). A sample of the thiocarbonate 7
(31.2 g, 35.5 mmol) and azobis-(cyclohexane-1-carbonitrile (ACCN, 2.86 g,

Evaluation of the Forodesine HCl Analog BCX-3040
1829
11.7 mmol, 0.33 eq.) was dissolved in anhydrous toluene (600 mL) under an
Argon atmosphere. The solution was sparged with argon for 30 min followed
by addition of trimethylsilane (23 mL, 74.5 mmol, 2.1 eq.). The resulting
solution was placed in a pre-heated 100◦C oil bath. After 1.5 hrs the solu-
tion was concentrated. The crude product was purified by chromatography
(Flash 75, 800 g SiO2, gradient 15 → 20% EtOAc-hexanes) and relevant frac-
tions combined to give 23.52 g (32.3 mmol, 91%) of 8 as a clear glass. IR
(KBr) 3054, 2947, 2868, 1687, 1610, 1535, and 1264 cm−1; MS (m/z, ES+)
1
727.3 (100%); H NMR (300 MHz, CDCl3) 8.50 (s, 1H), 7.40 (s, 1H),
7.34–7.23 (m, 5H), 5.75 (br d, J = 9.0 Hz, 1H), 5.57 (d, J = 10.5 Hz, 1H),
5.36 (d, J = 8.9 Hz, 1H), 4.96 (br s, 2H), 4.49–4.38 (m, 2H), 4.16–4.11 (m,
1H), 4.10 (s, 3H), 3.68–3.62 (m, 1H), 2.58 (br s, 1H), 2.42–2.31 (m, 1H),
1.40 (br s, 9H), 1.08–0.88 (m, 28H). Analysis calculated for C37H58N4O7Si2:
C, 61.12; H, 8.04; N, 7.71. Found: C, 61.46; H, 7.97; N, 7.72.
(1S)-N-tert-Butoxycarbonyl-1 -C-{4-methoxypyrrolo[3,2-d]pyrimidin-7-
yl}-1 ,4 -dideoxy-1 ,4 -imino-2 -deoxy-3 ,5 -O-(1,1,3,3-tetraisopropyldisiloxa-
1,3-diyl)-D-ribitol (9). A mixture of 8 (24.54 g, 33.8 mmol) and 20%
Pd(OH)2 on C (25 g) in EtOH (∼150 mL) containing concentrated
aqueous NH4OH (∼1 mL) was shaken under an H2 atmosphere (Parr
shaker, 40 psig) for 1.5 h. The mixture was filtered through Celite and the
pad washed with EtOH (75 mL). After the filtrate was concentrated the
residue was partitioned between dichloromethane and water. The organic
extract was washed with water, dried (Na2SO4), and concentrated to give
18.55 g (30.6 mmol, 90%) of 9 as a white foam. IR (KBr) 2947, 2868, 1697,
1
1675, 1627, 1537, and 1392 cm−1; MS (m/z, ES+) 607.2 (100%); H NMR
(300 MHz, CDCl3) 8.86 (br s, 1H, D2O exchangeable), 8.46 (br s, 1H),
7.26 (br s, 1H), 5.37 (d, J = 8.8 Hz, 1H), 4.89 (br s, 2H), 4.34–4.10 (m,
2H), 4.10 (s, 3H), 3.68–3.62 (m, 1H), 2.63 (br s, 1H), 2.40–2.30 (m, 1H),
1.44 (br s, 9H), 1.12–0.88 (m, 28H). Analysis calculated for C25H50N4O6Si2:
C, 57.39; H, 8.30; N, 9.23. Found: C, 57.39; H, 8.32; N, 9.22.
(1S)-2 -Deoxy-1 -C-{4-hydroxypyrrolo[3,2-d]pyrimidin-7-yl}-1 ,4 -imino-
D-ribitol Hydrochloride (1). A sample of 9 (18.05 g, 29.7 mmol) was dis-
solved in methanol (100 mL) under an Argon atmosphere. To this concen-
trated aqueous HCl (100 mL, ∼1200 mmol, ∼40 eq.) was added and the
mixture refluxed for 3 h. The solution was concentrated and the residue
taken up in a small amount of water (40 mL). Absolute EtOH was added and
the resulting mixture concentrated and the treatment with EtOH repeated
three times. The crude product was recrystallized from 1N HCl/EtOH to
give 7.05 g (24.6 mmol, 83%) of 1, m.p. 280◦C (dec). IR (KBr) 3472, 3422,
1
3118, 2845, 1683, 1660, and 1597 cm−1; MS (m/z, ES+) 251.0 (100%); H
NMR (300 MHz, DMSO-d6 and CDCl3) 12.37 (s, 1H, D2O exchangeable),
12.17 (br s, 1H, D2O exchangeable), 10.6–8.9 (br s, 1H, D2O exchangeable),

1830
H. S. Kezar III et al.
7.90 (s, 1H), 7.69 (s, 1H), 5.64 (d, J = 3.6 Hz, 1H, D2O exchangeable), 5.42
(br s, 1H, D2O exchangeable), 5.02 (dd, J = 6.2, 11.9 Hz, 1H), 4.35 (d, J =
2.0 Hz, 1H), 3.78–3.68 (m, 1H), 3.52–3.47 (m, 1H), 2.62–2.52 (m, 1H),
2.21 (dd, J = 6.6, 13.2 Hz, 1H). Analysis calculated for C11H14N4O3·HCl:
C, 46.08; H, 5.27; N, 19.54; Cl, 12.37. Found: C, 46.34; H, 5.30; N, 19.70;
Cl, 12.36.
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